Therapeutic DLL4 Binding Proteins

ABSTRACT

Improved DLL4 binding proteins are described, including antibodies, CDR-grafted antibodies, human antibodies, and DLL4 binding fragments thereof, proteins that bind DLL4 with high affinity, and DLL4 binding proteins that neutralize DLL4 activity. The DLL4 binding proteins are useful for treating or preventing cancers and tumors and especially for treating or preventing tumor angiogenesis, and/or other angiogenesis-dependent diseases such as ocular neovascularization, or angiogenesis-independent diseases characterized by aberrant DLL4 expression or activity such as autoimmune disorders including multiple sclerosis.

REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/295,149, filed Oct. 17, 2016 which is a Continuation of co-pendingU.S. patent application Ser. No. 14/822,329, filed Aug. 10, 2015, whichis a Division of U.S. patent application Ser. No. 14/090,778, filed Nov.26, 2013, now U.S. Pat. No. 9,132,190, issued Sep. 15, 2015, which is aDivision of U.S. patent application Ser. No. 12/870,276, filed Aug. 27,2010, now U.S. Pat. No. 8,623,358, issued Jan. 7, 2014, which claims thebenefit of priority to U.S. Provisional Application No. 61/261,728,filed Nov. 16, 2009, and U.S. Provisional Application No. 61/238,152,filed Aug. 29, 2009. The contents of each of the above prioritydocuments are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the development and use of improvedDLL4 binding proteins and uses thereof in the inhibition, prevention,and/or treatment of cancers, tumors, and/or other angiogenesis-dependentdiseases such as ocular neovascularization, or angiogenesis-independentdiseases characterized by aberrant DLL4 expression or activity such asautoimmune disorders.

BACKGROUND OF THE INVENTION

Cell-to-cell communication is required for many biological processessuch as differentiation, proliferation, and homeostasis. One systemutilized by a wide range of eukaryotes is the Notch-signaling pathway.This pathway, especially the Notch receptor, is also critical forfunctional tumor angiogenesis. Thus, inhibition of Notch receptorfunction, blockage of the Notch receptor, and/or blockage of theNotch-signaling pathway are potential strategies for anti-cancercompositions and therapies. Small molecule inhibitors of the Notchreceptor have proven to be toxic because they suppress wild type(normal) tissue expression of Notch receptors throughout the body. Thus,different members of the Notch-signaling pathway should be considered aspotential targets for therapeutics.

A vasculature ligand for the Notch receptor is Delta 4 or Delta-like 4(DLL4). Largely expressed in the vasculature. DLL4 is critical forvascular development (Yan et al., Clin. Cancer Res., 13(24): 7243-7246(2007); Shutter et al., Genes Dev., 14(11): 1313-1318 (2000); Gale etal., Proc. Natl. Acad Sci. USA, 101(45): 15949-15954 (2004); Krebs etal., Genes Dev., 14(11): 1343-1352 (2000)). Mice heterozygous for DLL4are embryonically lethal due to major defects in vascular development(Gale et al., Proc. Natl. Acad Sci. USA, 101(45): 15949-15954 (2004);Duarte et al., Genes Dev., 18(20): 2474-2478 (2004); Krebs et al., GenesDev., 18(20): 2469-2473 (2004)). The expression of DLL4 can be inducedby VEGF (Liu et al., Mol. Cell Biol., 23(1): 14-25 (2003); Lobov et al.,Proc. Natl. Acad Sci. USA, 104(9): 3219-3224 (2007)). In turn, DLL4 cannegatively regulate VEGF signaling, in part through repressing VEGFR2and inducing VEGFR1 (Harrington et al., Microvasc. Res., 75(2): 144-154(2008); Suchting et al., Proc. Natl. Acad Si. USA, 104(9): 3225-3230(2007)). Exquisite coordination between DLL4 and VEGF is essential forfunctional angiogenesis.

In addition to its physiological role. DLL4 is up-regulated in tumorblood vessels (Gale et al., Proc. Natl. Acad. Sci. USA, 101(45):15949-15954 (2004); Mailhos et al., Differentiation, 69(2-3): 135-144(2001); Patel et al., Cancer Res., 65(19): 8690-8697 (2005); Patel etal., Clin. Cancer Res., 12(16): 4836-4844 (2006): Noguera-Troise et al.,Nature, 444(7122): 1032-1037 (2006)). Blockade of DLL4 potentlyinhibited primary tumor growth in multiple models (Noguera-Troise etal., Nature, 444(7122): 1032-1037 (2006); Ridgway et al., Nature,444(7122): 1083-1087 (2006); Scehnet et al., Blood. 109(11): 4753-4760(2007)). The inhibition of DLL4 was even effective against tumors thatare resistant to anti-VEGF therapy. The combinatorial inhibition of bothDLL4 and VEGF provided an enhanced anti-tumor activity. Interestingly,unlike VEGF inhibition that reduces tumor vessel formation, DLL4blockade leads to an increase in tumor vasculature density wherein thevessels are abnormal, cannot support efficient blood transport, and areeffectively nonfunctional. Thus. DLL4 provides a potential target forcancer treatment.

There is a need in the art for therapeutic agents capable of targetingthe DLL4-Notch pathway and thereby inhibiting, or even preventing, tumorangiogenesis and growth.

SUMMARY OF THE INVENTION

The invention provides proteins that bind DLL4, including antibodies,CDR-grafted antibodies, and binding fragments thereof, that are capableof binding DLL4. Preferably, a binding protein described herein bindsDLL4 with high affinity. More preferably, a binding protein according tothe invention is capable of neutralizing DLL4. The invention alsoprovides methods of making and using DLL4 binding proteins, includinghuman DLL4 binding proteins. Advantageously, the invention eliminatesthe need to prepare humanized DLL4 binding proteins; thereby,eliminating the complications associated with humanized DLL4 bindingproteins.

One aspect of this invention pertains to a binding protein comprising anantigen binding domain capable of binding human DLL4, said antigenbinding domain comprising at least one or more CDRs selected from thegroup consisting of:

CDR-HL: (SEQ ID NO: 99) X₁-X₂-X₃-X₄-X₅-X₆-X₇,wherein;

-   -   X₁ is S or N;    -   X₂ is S, G, or N;    -   X₃ is S, N, T, G, or R;    -   X₄ is Y;    -   X₅ is Y or H;    -   X₆ is W; and

X₇ is G;

CDR-H2: (SEQ ID NO: 100)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X-₁₄-X₁₅-X₁₆,wherein;

-   -   X₁ is D;    -   X₂ is I;    -   X₃ is Y, N, or S;    -   X₄ is Y;    -   X₅ is T, N, A, I, S, or R;    -   X₆ is G;    -   X₇ is S, N, T, or G;    -   X₈ is T;    -   X₉ is Y;    -   X₁₀ is Y;    -   X₁₁ is N;    -   X₁₂ is P;    -   X₁₃ is S;    -   X₁₄ is L;    -   X₁₅ is K; and    -   X₁₆ is S, N, D, or G;

CDR-H3: (SEQ ID NO: 101) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁,

wherein;

-   -   X₁ is E, Y, F, Q, W, L, or A;    -   X₂ is D, A, S, G, V, E, or N;    -   X₃ is V, M, L, P, or A;    -   X₄ is I, A, P, R, S, K, Q, V, G, M, or E;    -   X₅ is L, Y, F, or M;    -   X₆ is R, G, S, Q, or A;    -   X₇ is G;    -   X₈ is G, A, or S;    -   X₉ is S, A, L, V, R, or G;    -   X₁₀ is D; and    -   X₁₁ is Y, D, S, N, H, E, R, L, P, C, I, M, T, Q,    -   or K;

CDR-L1: (SEQ ID NO: 102) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁,

wherein;

-   -   X₁ is S;    -   X₂ is G;    -   X₃ is Q, E, or D;    -   X₄ is R, S, G, M, K, L, or T;    -   X₅ is L;    -   X₆ is G;    -   X₇ is D or E;    -   X₈ is K;    -   X₉ is Y;    -   X₁₀ is A or V; and    -   X₁₁ is S;

CDRL2: (SEQ ID NO: 103) X₁-X₂-X₃-X₄-X₅-X₆-X₇,wherein

-   -   X₁ is E or Q;    -   X₂ is D;    -   X₃ is S, L, T, A, E, or F;    -   X₄ is K, T, E, N, Q, S, or M;    -   X₅ is R;    -   X₆ is P; and    -   X₇ is S;

CDR-L3: (SEQ ID NO: 104) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉,wherein;

-   -   X₁ is Q;    -   X₂ is A;    -   X₃ is W;    -   X₄ is D;    -   X₅ is R, S, M, E, N, G, or K;    -   X₆ is D or E;    -   X₇ is T, V, A, S, or M;    -   X₈ is G, A, or C; and    -   X₉ is V.

Preferably, the antigen binding domain of a DLL4 binding protein of theinvention comprises at least one CDR comprising an amino acid sequenceselected from the group consisting of:

-   -   residues 31-37 of SEQ ID NO:1 (CDR-H1); residues 52-67 of SEQ ID        NO:1 (CDR-H2); residues 100-110 of SEQ ID NO:1 (CDR-H3);    -   residues 23-33 of SEQ ID NO: 111 (CDR-L1); residues 49-55 of SEQ        ID NO: 111 (CDR-L2); residues 88-96 of SEQ ID NO: 111 (CDR-L3);    -   SEQ ID NO: 117 (CDR-H1): SEQ ID NO: 118 (CDR-H2); SEQ ID NO:119        (CDR-H3);    -   SEQ ID NO: 121 (CDR-H1); SEQ ID NO: 122 (CDR-H2); SEQ ID NO: 123        (CDR-H3);    -   SEQ ID NO:125 (CDR-H1); SEQ ID NO:126 (CDR-H2); SEQ ID NO:127        (CDR-H3);    -   SEQ ID NO:129 (CDR-H1); SEQ ID NO:130 (CDR-H2); SEQ ID NO:131        (CDR-H3);    -   SEQ ID NO: 133 (CDR-H1); SEQ ID NO:134 (CDR-H2); SEQ ID NO: 135        (CDR-H3);    -   SEQ ID NO:137 (CDR-H1); SEQ ID NO:138 (CDR-H2); SEQ ID NO:139        (CDR-H3);    -   SEQ ID NO:141 (CDR-H1); SEQ ID NO: 142 (CDR-H2); SEQ ID NO:143        (CDR-H3);    -   SEQ ID NO:145 (CDR-H1); SEQ ID NO:146 (CDR-H2); SEQ ID NO:147        (CDR-H3);    -   SEQ ID NO:149 (CDR-H1); SEQ ID NO:150 (CDR-H2); SEQ ID NO:151        (CDR-H3);    -   SEQ ID NO:153 (CDR-H1); SEQ ID NO:154 (CDR-H2); SEQ ID NO:155        (CDR-H3);    -   SEQ ID NO:157 (CDR-H1); SEQ ID NO:158 (CDR-H2); SEQ ID NO:159        (CDR-H3);    -   SEQ ID NO:161 (CDR-H1); SEQ ID NO:162 (CDR-H2); SEQ ID NO:163        (CDR-H3);    -   SEQ ID NO:165 (CDR-H1); SEQ ID NO:166 (CDR-H2); SEQ ID NO:167        (CDR-H3);    -   SEQ ID NO:169 (CDR-H1); SEQ ID NO:170 (CDR-H2); SEQ ID NO:171        (CDR-H3);    -   SEQ ID NO: 173 (CDR-H1); SEQ ID NO: 174 (CDR-H2); SEQ ID NO: 175        (CDR-H3);    -   SEQ ID NO:177 (CDR-H1); SEQ ID NO:178 (CDR-H2); SEQ ID NO:179        (CDR-H3);    -   SEQ ID NO: 181 (CDR-H1); SEQ ID NO: 182 (CDR-H2); SEQ ID NO: 183        (CDR-H3);    -   SEQ ID NO:185 (CDR-H1); SEQ ID NO:186 (CDR-H2); SEQ ID NO:187        (CDR-H3);    -   SEQ ID NO:189 (CDR-H1); SEQ ID NO:190 (CDR-H2); SEQ ID NO: 191        (CDR-H3);    -   SEQ ID NO:193 (CDR-H1); SEQ ID NO:194 (CDR-H2); SEQ ID NO:195        (CDR-H3);    -   SEQ ID NO:197 (CDR-H1); SEQ ID NO:198 (CDR-H2); SEQ ID NO: 199        (CDR-H3);    -   SEQ ID NO:201 (CDR-H1); SEQ ID NO:202 (CDR-H2); SEQ ID NO:203        (CDR-H3);    -   SEQ ID NO:205 (CDR-H1); SEQ ID NO:206 (CDR-H2); SEQ ID NO:207        (CDR-H3);    -   SEQ ID NO:209 (CDR-H1); SEQ ID NO:210 (CDR-H2); SEQ ID NO:211        (CDR-H3);    -   SEQ ID NO:213 (CDR-H1); SEQ ID NO:214 (CDR-H2); SEQ ID NO:215        (CDR-H3);    -   SEQ ID NO:217 (CDR-L1); SEQ ID NO:218 (CDR-L2); SEQ ID NO:219        (CDR-L3);    -   SEQ ID NO:221 (CDR-L1); SEQ ID NO:222 (CDR-L2); SEQ ID NO:223        (CDR-L3);    -   SEQ ID NO:225 (CDR-L); SEQ ID NO:226 (CDR-L2); SEQ ID NO:227        (CDR-L3);    -   SEQ ID NO:229 (CDR-L); SEQ ID NO:230 (CDR-L2); SEQ ID NO:231        (CDR-L3);    -   SEQ ID NO:233 (CDR-L1); SEQ ID NO:234 (CDR-L2); SEQ ID NO:235        (CDR-L3);    -   SEQ ID NO:237 (CDR-L1); SEQ ID NO:238 (CDR-L2); SEQ ID NO:239        (CDR-L3);    -   SEQ ID NO:241 (CDR-L1); SEQ ID NO:242 (CDR-L2); SEQ ID NO:243        (CDR-L3);    -   SEQ ID NO:245 (CDR-L1); SEQ ID NO:246 (CDR-L2); SEQ ID NO:247        (CDR-L3);    -   SEQ ID NO: 249 (CDR-L1); SEQ ID NO:250 (CDR-L2); SEQ ID NO:251        (CDR-L3);    -   SEQ ID NO:253 (CDR-L1); SEQ ID NO:254 (CDR-L2); SEQ ID NO:255        (CDR-L3);    -   SEQ ID NO:257 (CDR-L1); SEQ ID NO:258 (CDR-L2); SEQ ID NO:259        (CDR-L3);    -   SEQ ID NO: 261 (CDR-L1); SEQ ID NO:262 (CDR-L2); SEQ ID NO:263        (CDR-L3);    -   SEQ ID NO:265 (CDR-L1); SEQ ID NO:266 (CDR-L2); SEQ ID NO:267        (CDR-L3);    -   SEQ ID NO:269 (CDR-L); SEQ ID NO:270 (CDR-L2); SEQ ID NO:271        (CDR-L3);    -   SEQ ID NO:273 (CDR-L1): SEQ ID NO:274 (CDR-L2); SEQ ID NO:275        (CDR-L3);    -   SEQ ID NO:277 (CDR-L); SEQ ID NO:278 (CDR-L2); SEQ ID NO:279        (CDR-L3);    -   SEQ ID NO:281 (CDR-L1); SEQ ID NO:282 (CDR-L2); SEQ ID NO:283        (CDR-L3);    -   SEQ ID NO:285 (CDR-L1): SEQ ID NO:286 (CDR-L2); SEQ ID NO:287        (CDR-L3);    -   SEQ ID NO:289 (CDR-L1): SEQ ID NO:290 (CDR-L2); SEQ ID NO:291        (CDR-L3);    -   SEQ ID NO:293 (CDR-L1); SEQ ID NO:294 (CDR-L2); SEQ ID NO:295        (CDR-L3);    -   SEQ ID NO:297 (CDR-L): SEQ ID NO:298 (CDR-L2); SEQ ID NO:299        (CDR-L3);    -   SEQ ID NO:301 (CDR-L1): SEQ ID NO:302 (CDR-L2); SEQ ID NO:303        (CDR-L3);    -   SEQ ID NO:305 (CDR-L1); SEQ ID NO:306 (CDR-L2); SEQ ID NO:307        (CDR-L3);    -   SEQ ID NO:309 (CDR-L1); SEQ ID NO:310 (CDR-L2); SEQ ID NO:311        (CDR-L3);    -   SEQ ID NO:313 (CDR-L1): SEQ ID NO:314 (CDR-L2); SEQ ID NO:315        (CDR-L3);    -   residues 31-37 of SEQ ID NO:334 (CDR-H1); residues 52-67 of SEQ        ID NO:334 (CDR-H2); residues 100-110 of SEQ ID NO:334 (CDR-H3);    -   residues 23-33 of SEQ ID NO:335 (CDR-L1); residues 49-55 of SEQ        ID NO:335 (CDR-L2), residues 88-96 of SEQ ID NO:335 (CDR-L3);    -   residues 31-37 of SEQ ID NO:336 (CDR-H1); residues 52-67 of SEQ        ID NO:336 (CDR-H2); residues 100-110 of SEQ ID NO:336 (CDR-H3);    -   residues 23-33 of SEQ ID NO:337 (CDR-L1); residues 49-55 of SEQ        ID NO:337 (CDR-L2); residues 88-96 of SEQ ID NO:337 (CDR-L3);    -   residues 31-37 of SEQ ID NO:338 (CDR-H1); residues 52-67 of SEQ        ID NO:338 (CDR-H2); residues 100-110 of SEQ ID NO:338 (CDR-H3);    -   residues 23-33 of SEQ ID NO:339 (CDR-L1); residues 49-55 of SEQ        ID NO:339 (CDR-L2); residues 88-96 of SEQ ID NO:339 (CDR-L3);    -   residues 31-37 of SEQ ID NO:340 (CDR-H1); residues 52-67 of SEQ        ID NO:340 (CDR-H2); residues 100-110 of SEQ ID NO:340 (CDR-H3);    -   residues 23-33 of SEQ ID NO:341 (CDR-L1); residues 49-55 of SEQ        ID NO:341 (CDR-L2); residues 88-96 of SEQ ID NO:341 (CDR-L3);    -   residues 31-37 of SEQ ID NO:342 (CDR-H1); residues 52-67 of SEQ        ID NO:342 (CDR-H2); residues 100-110 of SEQ ID NO:342 (CDR-H3);    -   residues 23-33 of SEQ ID NO:343 (CDR-L1); residues 49-55 of SEQ        ID NO:343 (CDR-L2); residues 88-96 of SEQ ID NO:343 (CDR-L3);    -   residues 31-37 of SEQ ID NO:344 (CDR-H1); residues 52-67 of SEQ        ID NO:344 (CDR-H2); residues 100-110 of SEQ ID NO:344 (CDR-H3);    -   residues 24-34 of SEQ ID NO:345 (CDR-L1); residues 50-56 of SEQ        ID NO:345 (CDR-L2), residues 89-97 of SEQ ID NO:345 (CDR-L3);    -   residues 31-37 of SEQ ID NO:346 (CDR-H1); residues 52-67 of SEQ        ID NO:346 (CDR-H2); residues 100-110 of SEQ ID NO:346 (CDR-H3);    -   residues 23-33 of SEQ ID NO:347 (CDR-L1); residues 49-55 of SEQ        ID NO:347 (CDR-L2); residues 88-96 of SEQ ID NO:347 (CDR-L3);    -   residues 31-37 of SEQ ID NO:348 (CDR-H1); residues 52-67 of SEQ        ID NO:348 (CDR-H2); residues 100-110 of SEQ ID NO:348 (CDR-H3);    -   residues 24-34 of SEQ ID NO:349 (CDR-L1); residues 50-56 of SEQ        ID NO:349 (CDR-L2); residues 89-97 of SEQ ID NO:349 (CDR-L3);    -   residues 31-37 of SEQ ID NO:350 (CDR-H1); residues 52-67 of SEQ        ID NO:350 (CDR-H2); residues 100-110 of SEQ ID NO:350 (CDR-H3);    -   residues 24-34 of SEQ ID NO:351 (CDR-L1); residues 50-56 of SEQ        ID NO:351 (CDR-L2); residues 89-97 of SEQ ID NO:351 (CDR-L3);    -   residues 31-37 of SEQ ID NO:352 (CDR-H1); residues 52-67 of SEQ        ID NO:352 (CDR-H2), residues 100-110 of SEQ ID NO:352 (CDR-H3);    -   residues 24-34 of SEQ ID NO:353 (CDR-L1); residues 50-56 of SEQ        ID NO:353 (CDR-L2); residues 89-97 of SEQ ID NO:353 (CDR-L3);    -   residues 31-37 of SEQ ID NO:354 (CDR-H1); residues 52-67 of SEQ        ID NO:354 (CDR-H2); residues 100-110 of SEQ ID NO:354 (CDR-H3);    -   residues 24-34 of SEQ ID NO:355 (CDR-L1); residues 50-56 of SEQ        ID NO:355 (CDR-L2); residues 89-97 of SEQ ID NO:355 (CDR-L3);    -   residues 31-37 of SEQ ID NO:356 (CDR-H1); residues 52-67 of SEQ        ID NO:356 (CDR-H2); residues 100-110 of SEQ ID NO:356 (CDR-H3);    -   residues 23-33 of SEQ ID NO:357 (CDR-L1); residues 49-55 of SEQ        ID NO:357 (CDR-L2); residues 88-96 of SEQ ID NO:357 (CDR-L3);    -   residues 31-37 of SEQ ID NO:358 (CDR-H1); residues 52-67 of SEQ        ID NO:358 (CDR-H2); residues 100-110 of SEQ ID NO:358 (CDR-H3);    -   residues 23-33 of SEQ ID NO:359 (CDR-L1); residues 49-55 of SEQ        ID NO:359 (CDR-L2); residues 88-96 of SEQ ID NO:359 (CDR-L3);    -   residues 31-37 of SEQ ID NO:360 (CDR-H1); residues 52-67 of SEQ        ID NO:360 (CDR-H2); residues 100-110 of SEQ ID NO:360 (CDR-H3);    -   residues 23-33 of SEQ ID NO:361 (CDR-L1); residues 49-55 of SEQ        ID NO:361 (CDR-L2); residues 88-96 of SEQ ID NO:361 (CDR-L3).

In another embodiment, the binding protein comprises at least 3 CDRsdisclosed above.

Preferably, a DLL4 binding protein according to the invention comprisesone or more CDRs disclosed above. More preferably, the binding proteincomprises three or more CDRs disclosed above. Most preferably, a DLL4binding protein according to the invention comprises six CDRs describedabove, i.e., a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and aCDR-L3 described above.

In a preferred embodiment, the binding protein comprises at least 3 CDRsselected from the group consisting of the sequences disclosed above.

More preferably, the binding protein comprises 3 CDRs selected from aset of variable domain CDRs selected from the group below.

VH E9 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:1

CDR-H2: residues 52-67 of SEQ ID NO:1

CDR-H3 residues 100-110 of SEQ ID NO:1

VL E9 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:111

CDR-L2: residues 49-55 of SEQ ID NO: 111

CDR-L3: residues 88-96 of SEQ ID NO: 111

VH E9.4 CDR Set

CDR-H1: SEQ ID NO:117

CDR-H2: SEQ ID NO:118

CDR-H3: SEQ ID NO:119

VL E9.4 CDR Set

CDR-L1: SEQ ID NO:229

CDR-L2: SEQ ID NO:230

CDR-L3: SEQ ID NO: 231

VH E9.11 CDR Set

CDR-H1: SEQ ID NO:121

CDR-H2: SEQ ID NO: 122

CDR-H3: SEQ ID NO: 123

VL E9.11 CDR Set

CDR-L1: SEQ ID NO:233

CDR-L2: SEQ ID NO:234

CDR-L3: SEQ ID NO:235

VH E9.14 CDR Set

CDR-H1: SEQ ID NO:125

CDR-H2: SEQ ID NO: 126

CDR-H3: SEQ ID NO:127

VL E9.14 CDR Set

CDR-L1: SEQ ID NO:237

CDR-L2: SEQ ID NO:238

CDR-L3: SEQ ID NO:239

VH E9.17 CDR Set

CDR-H1: SEQ ID NO:129

CDR-H2: SEQ ID NO: 130

CDR-H3: SEQ ID NO: 131

VL E9.17 CDR Set

CDR-L1: SEQ ID NO:241

CDR-L2: SEQ ID NO:242

CDR-L3: SEQ ID NO:243

VH E9.18 CDR Set

CDR-H1: SEQ ID NO: 133

CDR-H2: SEQ ID NO: 134

CDR-H3: SEQ ID NO: 135

VL E9.18 CDR Set

CDR-L1: SEQ ID NO:245

CDR-L2: SEQ ID NO:246

CDR-L3: SEQ ID NO:247

VH E9.19 CDR Set

CDR-H1: SEQ ID NO:137

CDR-H2: SEQ ID NO: 138

CDR-H3: SEQ ID NO: 139

VL E9.19 CDR Set

CDR-L1: SEQ ID NO:249

CDR-L2: SEQ ID NO:250

CDR-L3: SEQ ID NO:251

VH E9.22 CDR Set

CDR-H1: SEQ ID NO:141

CDR-H2: SEQ ID NO: 142

CDR-H3: SEQ ID NO:143

VL E9.22 CDR Set

CDR-L1: SEQ ID NO:253

CDR-L2: SEQ ID NO:254

CDR-L3: SEQ ID NO:255

VH E9.48 CDR Set

CDR-H1: SEQ ID NO: 145

CDR-H2: SEQ ID NO:146

CDR-H3: SEQ ID NO: 147

VL E9.48 CDR Set

CDR-L1: SEQ ID NO:257

CDR-L2: SEQ ID NO:258

CDR-L3: SEQ ID NO: 259

VH E9.65 CDR Set

CDR-H1: SEQ ID NO:149

CDR-H2: SEQ ID NO:150

CDR-H3: SEQ ID NO: 151

VL E9.65 CDR Set

CDR-L1: SEQ ID NO:261

CDR-L2: SEQ ID NO:262

CDR-L3: SEQ ID NO:263

VH E9.66 CDR Set

CDR-H1: SEQ ID NO:153

CDR-H2: SEQ ID NO:154

CDR-H3: SEQ ID NO:155

VL E9.66 CDR Set

CDR-L1: SEQ ID NO:265

CDR-L2: SEQ ID NO:266

CDR-L3: SEQ ID NO: 267

VH E9.71 CDR Set

CDR-H1: SEQ ID NO:157

CDR-H2: SEQ ID NO: 158

CDR-H3: SEQ ID NO: 159

VL E9.71 CDR Set

CDR-L1: SEQ ID NO:269

CDR-L2: SEQ ID NO:270

CDR-L3: SEQ ID NO:271

VH E9.13 CDR Set

CDR-H1: SEQ ID NO:161

CDR-H2: SEQ ID NO: 162

CDR-H3: SEQ ID NO: 163

VL E9.13 CDR Set

CDR-L1: SEQ ID NO:217

CDR-L2: SEQ ID NO:218

CDR-L3: SEQ ID NO:219

VH E9.16 CDR Set

CDR-H1: SEQ ID NO:165

CDR-H2: SEQ ID NO: 166

CDR-H3: SEQ ID NO: 167

VL E9.16 CDR Set

CDR-L1: SEQ ID NO:221

CDR-L2: SEQ ID NO:222

CDR-L3: SEQ ID NO:223

VH E9.38 CDR Set

CDR-H1: SEQ ID NO: 169

CDR-H2: SEQ ID NO: 170

CDR-H3: SEQ ID NO:171

VL E9.38 CDR Set

CDR-L1: SEQ ID NO:225

CDR-L2: SEQ ID NO:226

CDR-L3: SEQ ID NO:227

VH E9.2B CDR Set

CDR-H1: SEQ ID NO:173

CDR-H2: SEQ ID NO:174

CDR-H3: SEQ ID NO: 175

VL E9.2B CDR Set

CDR-L1: SEQ ID NO:273

CDR-L2: SEQ ID NO:274

CDR-L3: SEQ ID NO:275

VH E9.1F CDR Set

CDR-H1: SEQ ID NO:177

CDR-H2: SEQ ID NO: 178

CDR-H3: SEQ ID NO:179

VL E9.1F CDR Set

CDR-L1: SEQ ID NO:277

CDR-L2: SEQ ID NO:278

CDR-L3: SEQ ID NO:279

VH E9.10H CDR Set

CDR-H1: SEQ ID NO:181

CDR-H2: SEQ ID NO:182

CDR-H3: SEQ ID NO: 183

VL E9.10H CDR Set

CDR-L1: SEQ ID NO:301

CDR-L2: SEQ ID NO:302

CDR-L3: SEQ ID NO:303

VH E9.5E CDR Set

CDR-H1: SEQ ID NO:185

CDR-H2: SEQ ID NO: 186

CDR-H3: SEQ ID NO: 187

VL E9.5E CDR Set

CDR-L1: SEQ ID NO:293

CDR-L2: SEQ ID NO:294

CDR-L3: SEQ ID NO:295

VH E9.10C CDR Set

CDR-H1: SEQ ID NO:189

CDR-H2: SEQ ID NO:190

CDR-H3: SEQ ID NO: 191

VL E9.10C CDR Set

CDR-L1: SEQ ID NO:281

CDR-L2: SEQ ID NO:282

CDR-L3: SEQ ID NO: 283

VH E9.7E CDR Set

CDR-H1: SEQ ID NO: 193

CDR-H2: SEQ ID NO: 194

CDR-H3: SEQ ID NO: 195

VL E9.7E CDR Set

CDR-L1: SEQ ID NO:289

CDR-L2: SEQ ID NO:290

CDR-L3: SEQ ID NO:291

VH E9.12B CDR Set

CDR-H1: SEQ ID NO:197

CDR-H2: SEQ ID NO: 198

CDR-H3: SEQ ID NO: 199

VL E9.12B CDR Set

CDR-L1: SEQ ID NO:297

CDR-L2: SEQ ID NO:298

CDR-L3: SEQ ID NO:299

VH E9.10E CDR Set

CDR-H1: SEQ ID NO:201

CDR-H2: SEQ ID NO:202

CDR-H3: SEQ ID NO:203

VL E9.10E CDR Set

CDR-L1: SEQ ID NO:285

CDR-L2: SEQ ID NO:286

CDR-L3: SEQ ID NO:287

VH E9.6A CDR Set

CDR-H1: SEQ ID NO:205

CDR-H2: SEQ ID NO:206

CDR-H3: SEQ ID NO:207

VL E9.6A CDR Set

CDR-L1: SEQ ID NO:305

CDR-L2: SEQ ID NO:306

CDR-L3: SEQ ID NO:307

VH E9.7A CDR Set

CDR-H1: SEQ ID NO:209

CDR-H2: SEQ ID NO:210

CDR-H3: SEQ ID NO:211

VL E9.7A CDR Set

CDR-L1: SEQ ID NO:309

CDR-L2: SEQ ID NO:310

CDR-L3: SEQ ID NO:311

VH E9.8H CDR Set

CDR-H1: SEQ ID NO:213

CDR-H2: SEQ ID NO:214

CDR-H3: SEQ ID NO:215

VL E9.8H CDR Set

CDR-L1: SEQ ID NO:313

CDR-L2: SEQ ID NO:314

CDR-L3: SEQ ID NO:315

VH E9.1 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:334

CDR-H2: residues 52-67 of SEQ ID NO:334

CDR-H3: residues 100-110 of SEQ ID NO:334

VL E9.1 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:335

CDR-L2: residues 49-55 of SEQ ID NO:335

CDR-L3: residues 88-96 of SEQ ID NO:335

VH E9-SE1 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:336

CDR-H2: residues 52-67 of SEQ ID NO:336

CDR-H3: residues 100-110 of SEQ ID NO:336

VL E9-SE1 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:337

CDR-L2: residues 49-55 of SEQ ID NO:337

CDR-L3: residues 88-96 of SEQ ID NO:337

VH E9-SE2 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:338

CDR-H2: residues 52-67 of SEQ ID NO:338

CDR-H3: residues 100-110 of SEQ ID NO:338

VL E9-SE2 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:339

CDR-L2: residues 49-55 of SEQ ID NO:339

CDR-L3: residues 88-96 of SEQ ID NO:339

VH E9-SE3 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:340

CDR-H2: residues 52-67 of SEQ ID NO:340

CDR-H3: residues 100-110 of SEQ ID NO:340

VL E9-SE3 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:341

CDR-L2: residues 49-55 of SEQ ID NO:341

CDR-L3: residues 88-96 of SEQ ID NO:341

VH E9-SE4 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:342

CDR-H2: residues 52-67 of SEQ ID NO:342

CDR-H3: residues 100-110 of SEQ ID NO:342

VL E9-SE4 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:343

CDR-L2: residues 49-55 of SEQ ID NO:343

CDR-L3: residues 88-96 of SEQ ID NO:343

VH E9-SE5 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:344

CDR-H2: residues 52-67 of SEQ ID NO:344

CDR-H3: residues 100-110 of SEQ ID NO:344

VL E9-SE5 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:345

CDR-L2: residues 50-56 of SEQ ID NO:345

CDR-L3: residues 89-97 of SEQ ID NO:345

VH E9-SE6 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:346

CDR-H2: residues 52-67 of SEQ ID NO:346

CDR-H3: residues 100-110 of SEQ ID NO:346

VL E9-SE6 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:347

CDR-L2: residues 49-55 of SEQ ID NO:347

CDR-L3: residues 88-96 of SEQ ID NO:347

VH E9-SE7 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:348

CDR-H2: residues 52-67 of SEQ ID NO:348

CDR-H3: residues 100-110 of SEQ ID NO:348

VL E9-SE7 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:349

CDR-L2: residues 50-56 of SEQ ID NO:349

CDR-L3: residues 89-97 of SEQ ID NO:349

VH E9-SE8 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:350

CDR-H2: residues 52-67 of SEQ ID NO:350

CDR-H3: residues 100-110 of SEQ ID NO:350

VL E9-SE8 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:351

CDR-L2: residues 50-56 of SEQ ID NO:351

CDR-L3: residues 89-97 of SEQ ID NO:351

VH E9-FR1 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:352

CDR-H2: residues 52-67 of SEQ ID NO:352

CDR-H3: residues 100-110 of SEQ ID NO:352

VL E9-FR1 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:353

CDR-L2: residues 50-56 of SEQ ID NO:353

CDR-L3: residues 89-97 of SEQ ID NO:353

VH E9-FR2 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:354

CDR-H2: residues 52-67 of SEQ ID NO:354

CDR-H3: residues 100-110 of SEQ ID NO:354

VL E9-FR2 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:355

CDR-L2: residues 50-56 of SEQ ID NO:355

CDR-L3: residues 89-97 of SEQ ID NO:355

VH E9.71 CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:356

CDR-H2: residues 52-67 of SEQ ID NO:356

CDR-H3: residues 100-110 of SEQ ID NO:356

VL E9.71 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:357

CDR-L2: residues 49-55 of SEQ ID NO:357

CDR-L3: residues 88-96 of SEQ ID NO:357

VH E9.71(M) CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:358

CDR-H2: residues 52-67 of SEQ ID NO:358

CDR-H3: residues 100-110 of SEQ ID NO:358

VL E9.71(M) CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:359

CDR-L2: residues 49-55 of SEQ ID NO:359

CDR-L3: residues 88-96 of SEQ ID NO:359

VH E9.71(L) CDR Set

CDR-H1: residues 31-37 of SEQ ID NO:360

CDR-H2: residues 52-67 of SEQ ID NO:360

CDR-H3: residues 100-110 of SEQ ID NO:360

VL E9.71(L) CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:361

CDR-L2: residues 49-55 of SEQ ID NO:361

CDR-L3: residues 88-96 of SEQ ID NO:361

In another embodiment, a binding protein comprises a variable heavychain (VH) set of 3 CDRs selected from any VH set of 3 CDRs in the groupabove and a variable light chain (VL) set of 3 CDRS selected from any VLset of 3 CDRs in the group above.

In still another embodiment a binding protein comprises a named VH setof 3 CDRs and a correspondingly named VL set of 3 CDRs from the groupbelow. Preferably, a binding protein according to the inventioncomprises at least two variable domain CDR sets selected from the groupof variable domain CDR sets consisting of:

VH E9 CDR Set and VL E9 CDR Set,

VH E9.4 CDR Set and VL E9.4 CDR Set.

VH E9.11 CDR Set and VL E9.11 CDR Set,

VH E9.14 CDR Set and VL E9.14 CDR Set.

VH E9.17 CDR Set and VL E9.17 CDR Set,

VH E9.18 CDR Set and VL E9.18 CDR Set,

VH E9.19 CDR Set and VL E9.19 CDR Set,

VH E9.22 CDR Set and VL E9.22 CDR Set,

VH E9.48 CDR Set and VL E9.48 CDR Set,

VH E9.65 CDR Set and VL E9.65 CDR Set.

VH E9.66 CDR Set and VL E9.66 CDR Set.

VH E9.71 CDR Set and VL E9.71 CDR Set,

VH E9.13 CDR Set and VL E9.13 CDR Set,

VH E9.16 CDR Set and VL E9.16 CDR Set.

VH E9.38 CDR Set and VL E9.38 CDR Set,

VH E9.2B CDR Set and VL E9.2B CDR Set,

VH E9.1F CDR Set and VL E9.1F CDR Set,

VH E9.10H CDR Set and VL E9.10H CDR Set,

VH E9.5E CDR Set and VL E9.5E CDR Set,

VH E9.10C CDR Set and VL E9.10C CDR Set,

VH E9.7E CDR Set and VL E9.7E CDR Set,

VH E9.12B CDR Set and VL E9.12B CDR Set,

VH E9.10E CDR Set and VL E9.10E CDR Set.

VH E9.6A CDR Set and VL E9.6A CDR Set,

VH E9.7A CDR Set and VL E9.7A CDR Set,

VH E9.8H CDR Set and VL E9.8H CDR Set.

VH E9-SE1 CDR Set and VL E9-SE1 CDR Set

VH E9-SE2 CDR Set and VL E9-SE2 CDR Set,

VH E9-SE3 CDR Set and VL E9-SE3 CDR Set,

VH E9-SE4 CDR Set and VL E9-SE4 CDR Set,

VH E9-SE5 CDR Set and VL E9-SE5 CDR Set,

VH E9-SE6 CDR Set and VL E9-SE6 CDR Set,

VH E9-SE7 CDR Set and VL E9-SE7 CDR Set,

VH E9-SE8 CDR Set and VL E9-SE8 CDR Set.

VH E9-FR1 CDR Set and VL E9-FR1 CDR Set.

VH E9-FR2 CDR Set and VL E9-FR2 CDR Set.

VH E9.71 CDR Set and VL E9.71 CDR Set.

VH E9.71(M) CDR Set and VL E9.71(M) CDR Set, and

VH E9.71(L) CDR Set and VL E9.71(L) CDR Set.

In yet another embodiment, a binding protein described above furthercomprises a human acceptor framework. Preferably, the human acceptorframework comprises an amino acid sequence selected from the groupconsisting of:

-   -   heavy chain acceptor framework sequences SEQ ID NOS:6-22.    -   heavy chain acceptor sequences SEQ ID NOS:35-62.    -   light chain acceptor sequences SEQ ID NOS:23-34, and    -   light chain acceptor sequences SEQ ID NOS:63-98.

In another embodiment a binding protein described above comprises ahuman acceptor framework, which comprises at least one framework regionamino acid substitution, wherein the amino acid sequence of theframework is at least 65% identical to the sequence of said humanacceptor framework and comprising at least 70 amino acid residuesidentical to said human acceptor framework.

In another embodiment a binding protein described herein comprises ahuman acceptor framework that comprises at least one framework regionamino acid substitution at a key residue, said key residue selected fromthe group consisting of:

-   -   a residue adjacent to a CDR;    -   a glycosylation site residue;    -   a rare residue;    -   a residue capable of interacting with human DLL4    -   a canonical residue;    -   a contact residue between heavy chain variable region and light        chain variable region;    -   a residue within a Vernier zone; and    -   a residue in a region that overlaps between a Chothia-defined        variable heavy    -   chain CDR1 and a Kabat-defined first heavy chain framework.        Preferably, the key residue is selected from the group        consisting of: 2H, 4H, 24H, 26H, 27H, 29H, 34H, 35H, 37H, 39H,        44H, 45H, 47H, 48H, 49H, 50H51H, 58H, 59H, 60H, 63H, 67H, 69H,        71H, 73H, 76H, 78H, 91H, 93H, 94H, 2L, 4L, 25L, 29L, 27bL, 33L,        34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L, 49L, 55L, 58L, 62L, 64L,        71L, 87L, 89L, 90L, 91L, 94L, 95L.

In another embodiment, a binding protein described herein comprises aconsensus human variable domain.

In a preferred embodiment, a binding protein described above comprisesat least one variable domain having amino acid sequence selected fromthe group consisting of: SEQ ID NOS:1, 111, 116, 228, 120, 232, 124,236, 128, 240, 132, 244, 136, 248, 140, 252, 144, 256, 148, 260, 152,264, 156, 268, 160, 216, 164, 220, 168, 224, 172, 272, 176, 276, 180,300, 184, 292, 188, 280, 192, 288, 196, 296, 200, 284, 204, 304, 208,308, 212, 312, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, and 361.

In another embodiment, a binding protein according to the inventioncomprises two variable domains, wherein said two variable domains haveamino acid sequences selected from the group consisting of: SEQ ID NOS:1and 111, SEQ ID NOS: 116 and 228, SEQ ID NOS: 120 and 232, SEQ IDNOS:124 and 236, SEQ ID NOS:128 and 240, SEQ ID NOS:132 and 244, SEQ IDNOS:136 and 248, SEQ ID NOS:140 and 252, SEQ ID NOS:144 and 256, SEQ IDNOS:148 and 260, SEQ ID NOS:152 and 264, SEQ ID NOS:156 and 268, SEQ IDNOS: 160 and 216, SEQ ID NOS: 164 and 220, SEQ ID NOS:168 and 224, SEQID NOS:172 and 272, SEQ ID NOS:176 and 276, SEQ ID NOS:180 and 300, SEQID NOS:184 and 292, SEQ ID NOS:188 and 280, SEQ ID NOS:192 and 288, SEQID NOS:196 and 296, SEQ ID NOS:200 and 284, SEQ ID NOS:204 and 304, SEQID NOS:208 and 308, SEQ ID NOS:212 and 312, SEQ ID NOS:334 and 335, SEQID NOS:336 and 337, SEQ ID NOS:338 and 339, SEQ ID NOS:340 and 341, SEQID NOS:342 and 343, SEQ ID NOS:344 and 345, SEQ ID NOS:346 and 347, SEQID NOS:348 and 349, SEQ ID NOS:350 and 351, SEQ ID NOS:352 and 353, SEQID NOS:354 and 355, SEQ ID NOS:356 and 357, SEQ ID NOS:358 and 359, SEQID NOS:360 and 361.

In an embodiment, a binding protein according to the invention comprisesheavy chain variable domain (V_(H)), preferably wherein the V_(H)comprises an amino acid sequence selected from the group consisting of:

SEQ ID NOS:1, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156,160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, and360.

In yet another embodiment, a binding protein according to the inventioncomprises a light chain variable domain (a V_(L)), preferably whereinthe V_(L) comprises an amino acid sequence selected from the groupconsisting of:

SEQ ID NOS: 111, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268,216, 220, 224, 272, 276, 300, 292, 280, 288, 296, 284, 304, 308, 312,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, and361.

In a preferred embodiment, a binding protein according to the inventioncomprises a V_(H) and a V_(L), preferably wherein V_(H) and a V_(L) areany of those sequences disclosed above.

In another embodiment, the invention provides a binding protein capableof binding human DLL-4, said binding protein comprising:

an Ig constant heavy region having an amino acid sequence selected fromthe group consisting of SEQ ID NO:2 and SEQ ID NO:3;

an Ig constant light region having an amino acid sequence selected fromthe group consisting of SEQ ID NO:4 and SEQ ID NO:5;

an Ig variable heavy region having an amino acid sequence selected fromthe group consisting:

SEQ ID NOS:1, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156,160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, and360; and

an Ig variable light region having an amino acid sequence selected fromthe group consisting:

SEQ ID NOS: 111, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268,216, 220, 224, 272, 276, 300, 292, 280, 288, 296, 284, 304, 308, 312,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, and361.

Another aspect of the invention pertains to a binding protein comprisingan antigen binding domain capable of binding human DLL4, said antigenbinding domain comprising at least one or more CDRs selected from thegroup consisting of:

(SEQ ID NO: 105) CDR-H1: X₁-X₂-X₃-X₄-X_(5,)wherein;

-   -   X₁ is S, N, or D;    -   X₂ is H or Y;    -   X₃ is W;    -   X₄ is M;    -   X₅ is S or H;

(SEQ ID NO: 106) CDR-H2: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X_(17,)wherein;

-   -   X₁ is I, D, M, or T;    -   X₂ is I;    -   X₃ is S;    -   X₄ is Y, N, S, Q, V, T, H, or D;    -   X₅ is D;    -   X₆ is G;    -   X₇ is S, R, I, T, G, K, H, or N;    -   X₈ is N, Y, S, I, or T;    -   X₉ is K, M, N, Q, E, T, R, S, A, or L;    -   X₁₀ is Y, D, or E;    -   X₁₁ is S or Y;    -   X₁₂ is A;    -   X₁₃ is D;    -   X₁₄ is S;    -   X₁₅ is V;    -   X₁₆ is K; and    -   X₁₇ is G;

(SEQ ID NO: 107) CDR-H3: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X_(10,)

wherein;

-   -   X₁ is A;    -   X₂ is G, A, or R;    -   X₃ is G;    -   X₄ is G, S, or A;    -   X₅ is N;    -   X₆ is V or M;    -   X₇ is G;    -   X₈ is F, L, Y, or M;    -   X₉ is D; and    -   X₁₀ is I, S, or L;

(SEQ ID NO: 108) CDR-L1: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X_(11,)

wherein;

-   -   X₁ is S;    -   X₂ is A or G;    -   X₃ is D;    -   X₄ is K, N, L, Q, M, E, S, T, G, or D;    -   X₅ is L;    -   X₆ is G;    -   X₇ is T, S, N, A, G, or E;    -   X₈ is K, Q, N, or R;    -   X₉ is Y;    -   X₁₀ is V or I; and    -   X₁₁ is S;

(SEQ ID NO: 109) CDR-L2: X₁-X₂-X₃-X₄-X₅-X₆-X_(7,)wherein;

-   -   X₁ is Q;    -   X₂ is D;    -   X₃ is A, G, W, S, or D;    -   X₄ is K, M, Q, N, L, T, I, or E;    -   X₅ is R;    -   X₆ is P; and    -   X₇ is S;

and

(SEQ ID NO: 110) CDR-L3: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X_(9,)wherein;

-   -   X₁ is Q;    -   X₂ is S or A;    -   X₃ is W;    -   X₄ is D;    -   X₅ is R, S, Q, P, A, V, W, or M;    -   X₆ is S, G, I, N, R, or T;    -   X₇ is D or G;    -   X₈ is V, A, P, or E; and    -   X₉ is V.

Preferably, the antigen binding domain of a DLL4 binding protein of theinvention comprises at least one CDR comprising an amino acid sequenceselected from the group consisting of:

-   -   residues 31-35 of SEQ ID NO:112 (CDR-H1); residues 50-66 of SEQ        ID NO:112 (CDR-H2); residues 99-108 of SEQ ID NO: 112 (CDR-H3);    -   residues 23-33 of SEQ ID NO: 113 (CDR-L1); residues 49-55 of SEQ        ID NO: 113 (CDR-L2); residues 88-96 of SEQ ID NO: 113 (CDR-L3);    -   residues 31-35 of SEQ ID NO:316 (CDR-H1); residues 50-66 of SEQ        ID NO:316 (CDR-H2); residues 99-108 of SEQ ID NO:316 (CDR-H3);    -   residues 31-35 of SEQ ID NO:317 (CDR-H1); residues 50-66 of SEQ        ID NO:317 (CDR-H2); residues 99-108 of SEQ ID NO:317 (CDR-H3);    -   residues 31-35 of SEQ ID NO:318 (CDR-H1); residues 50-66 of SEQ        ID NO:318 (CDR-H2); residues 99-108 of SEQ ID NO:318 (CDR-H3);    -   residues 31-35 of SEQ ID NO:319 (CDR-H1); residues 50-66 of SEQ        ID NO:319 (CDR-H2); residues 99-108 of SEQ ID NO:319 (CDR-H3);    -   residues 31-35 of SEQ ID NO:320 (CDR-H1); residues 50-66 of SEQ        ID NO:320 (CDR-H2); residues 99-108 of SEQ ID NO:320 (CDR-H3);    -   residues 31-35 of SEQ ID NO:321 (CDR-H1); residues 50-66 of SEQ        ID NO:321 (CDR-H2); residues 99-108 of SEQ ID NO:321 (CDR-H3);    -   residues 31-35 of SEQ ID NO:322 (CDR-H1); residues 50-66 of SEQ        ID NO:322 (CDR-H2); residues 99-108 of SEQ ID NO:322 (CDR-H3);    -   residues 31-35 of SEQ ID NO:323 (CDR-H1); residues 50-66 of SEQ        ID NO:323 (CDR-H2); residues 99-108 of SEQ ID NO:323 (CDR-H3);    -   residues 31-35 of SEQ ID NO:324 (CDR-H1); residues 50-66 of SEQ        ID NO:324 (CDR-H2); residues 99-108 of SEQ ID NO:324 (CDR-H3);    -   residues 31-35 of SEQ ID NO:325 (CDR-H1); residues 50-66 of SEQ        ID NO:325 (CDR-H2); residues 99-108 of SEQ ID NO:325 (CDR-H3);    -   residues 31-35 of SEQ ID NO:326 (CDR-H1); residues 50-66 of SEQ        ID NO:326 (CDR-H2); residues 99-108 of SEQ ID NO:326 (CDR-H3);    -   residues 23-33 of SEQ ID NO:327 (CDR-L1); residues 49-55 of SEQ        ID NO:327 (CDR-L2); residues 88-96 of SEQ ID NO:327 (CDR-L3);    -   residues 23-33 of SEQ ID NO:328 (CDR-L1); residues 49-55 of SEQ        ID NO:328 (CDR-L2) residues 88-96 of SEQ ID NO:328 (CDR-L3);    -   residues 23-33 of SEQ ID NO:329 (CDR-L1); residues 49-55 of SEQ        ID NO:329 (CDR-L2); residues 88-96 of SEQ ID NO:329 (CDR-L3);    -   residues 23-33 of SEQ ID NO:330 (CDR-L1); residues 49-55 of SEQ        ID NO:330 (CDR-L2); residues 88-96 of SEQ ID NO:330 (CDR-L3);    -   residues 23-33 of SEQ ID NO:331 (CDR-L1); residues 49-55 of SEQ        ID NO:331 (CDR-L2); residues 88-96 of SEQ ID NO:331 (CDR-L3);    -   residues 23-33 of SEQ ID NO:332 (CDR-L1); residues 49-55 of SEQ        ID NO:332 (CDR-L2); residues 88-96 of SEQ ID NO:332 (CDR-L3);    -   residues 23-33 of SEQ ID NO:333 (CDR-L1); residues 49-55 of SEQ        ID NO:333 (CDR-L2) residues 88-96 of SEQ ID NO:333 (CDR-L3).

In another embodiment, the binding protein comprises at least 3 CDRsdisclosed above.

Preferably, a DLL4 binding protein according to the invention comprisesone or more CDRs disclosed above. More preferably, the binding proteincomprises three or more CDRs disclosed above. Most preferably, a DLL4binding protein according to the invention comprises six CDRs describedabove, i.e., a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and aCDR-L3 described above.

In a preferred embodiment, the binding protein comprises at least 3 CDRsselected from the group consisting of the sequences disclosed above.

In another preferred embodiment, a binding protein comprises 3 CDRsselected from a set of variable domain CDRs selected from the groupbelow.

VH A10 CDR St

CDR-H1: residues 31-35 of SEQ ID NO:112

CDR-H2: residues 50-66 of SEQ ID NO:112

CDR-H3: residues 99-108 of SEQ ID NO:112

VL A10 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:113

CDR-L2: residues 49-55 of SEQ ID NO: 113

CDR-L3: residues 88-96 of SEQ ID NO: 113

VH A10.3 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:316

CDR-H2: residues 50-66 of SEQ ID NO:316

CDR-H3: residues 99-108 of SEQ ID NO:316

VL A10.3 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:327

CDR-L2: residues 49-55 of SEQ ID NO:327

CDR-L3: residues 88-96 of SEQ ID NO:327

VH A10.K30 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:317

CDR-H2: residues 50-66 of SEQ ID NO:317

CDR-H3: residues 99-108 of SEQ ID NO:317

VH A10.K42 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:318

CDR-H2: residues 50-66 of SEQ ID NO:318

CDR-H3: residues 99-108 of SEQ ID NO:318

VH A10.9A CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:319

CDR-H2: residues 50-66 of SEQ ID NO:319

CDR-H3: residues 99-108 of SEQ ID NO:319

VH A10.8A CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:320

CDR-H2: residues 50-66 of SEQ ID NO:320

CDR-H3: residues 99-108 of SEQ ID NO:320

VH A10.1A CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:321

CDR-H2: residues 50-66 of SEQ ID NO:321

CDR-H3: residues 99-108 of SEQ ID NO:321

VH A10.5D CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:322

CDR-H2: residues 50-66 of SEQ ID NO:322

CDR-H3: residues 99-108 of SEQ ID NO:322

VH A10.3A CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:323

CDR-H2: residues 50-66 of SEQ ID NO:323

CDR-H3: residues 99-108 of SEQ ID NO:323

VL A10.3A CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:330

CDR-L2: residues 49-55 of SEQ ID NO:330

CDR-L3: residues 88-96 of SEQ ID NO:330

VH A10.6B CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:324

CDR-H2: residues 50-66 of SEQ ID NO:324

CDR-H3: residues 99-108 of SEQ ID NO:324

VL A10.6B CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:331

CDR-L2: residues 49-55 of SEQ ID NO:331

CDR-L3: residues 88-96 of SEQ ID NO:331

VH A10.3D CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:325

CDR-H2: residues 50-66 of SEQ ID NO:325

CDR-H3: residues 99-108 of SEQ ID NO:325

VL A10.3D CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:332

CDR-L2: residues 49-55 of SEQ ID NO:332

CDR-L3: residues 88-96 of SEQ ID NO:332

VH A10.4C CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:326

CDR-H2: residues 50-66 of SEQ ID NO:326

CDR-H3: residues 99-108 of SEQ ID NO:326

VL A10.4C CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:333

CDR-L2: residues 49-55 of SEQ ID NO:333

CDR-L3: residues 88-96 of SEQ ID NO:333

VL A10.L45 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:328

CDR-L2: residues 49-55 of SEQ ID NO:328

CDR-L3: residues 88-96 of SEQ ID NO:328

VL A10.L73 CDR Set

CDR-L1: residues 23-33 of SEQ ID NO:329

CDR-L2: residues 49-55 of SEQ ID NO:329

CDR-L3: residues 88-96 of SEQ ID NO:329

In another embodiment, a binding protein comprises a variable heavychain (VH) set of 3 CDRs selected from any VH set of 3 CDRs in the groupabove and a variable light chain (VL) set of 3 CDRS selected from any VLset of 3 CDRs in the group above.

In still another embodiment a binding protein comprises a named VH setof 3 CDRs and a correspondingly named VL set of 3 CDRs from the groupbelow. Preferably, a binding protein according to the inventioncomprises at least two variable domain CDR sets selected from the groupof variable domain CDR sets consisting of:

-   -   VH A10 CDR Set and VL A10 CDR Set;    -   VH A10.3 CDR Set and VL A10.3 CDR Set;    -   VH A10.3A CDR Set and VL A10.3A Set;    -   VH A10.6B CDR Set and VL A10.6B Set;    -   VH A10.3D CDR Set and VL A10.3D CDR Set;    -   VH A10.4C CDR Set and VL A10.4C CDR Set.    -   VH A10.K30 CDR Set and VL A10.3 CDR Set;    -   VH A10.K42 CDR Set and VL A10.3 CDR Set;    -   VH A10.3 CDR Set and VL A10.L45 CDR Set;    -   VH A10.3 CDR Set and VL A10.L73 CDR Set;    -   VH A10.9A CDR Set and VL A10.3 CDR Set;    -   VH A10.8A CDR Set and VL A10.3 CDR Set;    -   VH A10.1A CDR Set and VL A10.3 CDR Set; and    -   VH A10.5D CDR Set and VL A10.3 CDR Set.

In yet another embodiment, a binding protein described above furthercomprises a human acceptor framework. Preferably, the human acceptorframework comprises an amino acid sequence selected from the groupconsisting of:

-   -   heavy chain acceptor framework sequences SEQ ID NOS:6-22.    -   heavy chain acceptor sequences SEQ ID NOS:35-62,    -   light chain acceptor sequences SEQ ID NOS:23-34, and    -   light chain acceptor sequences SEQ ID NOS:63-98.

In another embodiment a binding protein described above comprises ahuman acceptor framework, which comprises at least one framework regionamino acid substitution, wherein the amino acid sequence of theframework is at least 65% identical to the sequence of said humanacceptor framework and comprising at least 70 amino acid residuesidentical to said human acceptor framework.

In another embodiment a binding protein described herein comprises ahuman acceptor framework that comprises at least one framework regionamino acid substitution at a key residue, said key residue selected fromthe group consisting of:

-   -   a residue adjacent to a CDR    -   a glycosylation site residue;    -   a rare residue;    -   a residue capable of interacting with human DLL4    -   a canonical residue;    -   a contact residue between heavy chain variable region and light        chain variable region;    -   a residue within a Vernier zone; and    -   a residue in a region that overlaps between a Chothia-defined        variable heavy chain CDR1 and a Kabat-defined first heavy chain        framework.        Preferably, the key residue is selected from the group        consisting of: 2H, 4H, 24H, 26H, 27H, 29H, 34H, 35H, 37H, 39H,        44H, 45H, 47H, 48H, 49H, 50H, 51H, 58H, 59H, 60H, 63H, 67H, 69H,        71H, 73H, 76H, 78H, 91H, 93H, 94H, 2L, 4L, 25L, 29L, 27bL, 33L,        34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L, 49L, 55L, 58L, 62L, 64L,        71L, 87L, 89L, 90L, 91L, 94L, 95L.

In another embodiment, a binding protein described herein comprises aconsensus human variable domain.

In a preferred embodiment, a binding protein described above comprisesat least one variable domain having amino acid sequence selected fromthe group consisting of: SEQ ID NOS:112, 113, 316, 327, 317, 318, 319,320, 321, 322, 323, 330, 324, 331, 325, 332, 326, 333, 328, and 329.

In another embodiment, a binding protein described above comprises twovariable domains, wherein said two variable domains have amino acidsequences selected from the group consisting of: SEQ ID NOS:112 and 113.SEQ ID NOS:316 and 327. SEQ ID NOS:323 and 330, SEQ ID NOS:324 and 331,SEQ ID NOS:325 and 332, and SEQ ID NOS:326 and 333.

In an embodiment, a binding protein according to the invention comprisesheavy chain variable domain (V_(H)), preferably wherein the V_(H)comprises an amino acid sequence selected from the group consisting of:

SEQ ID NOS:112, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, and326.

In yet another embodiment, a binding protein according to the inventioncomprises a light chain variable domain (a V_(L)), preferably whereinthe V_(L) comprises an amino acid sequence selected from the groupconsisting of:

SEQ ID NOS: 113, 327, 328, 329, 330, 331, 332, and 333.

In another embodiment, the invention provides a binding protein capableof binding human DLL-4, said binding protein comprising:

an Ig constant heavy region having an amino acid sequence selected fromthe group consisting of SEQ ID NO:2 and SEQ ID NO:3;

an Ig constant light region having an amino acid sequence selected fromthe group consisting of SEQ ID NO:4 and SEQ ID NO:5;

an Ig variable heavy region having an amino acid sequence selected fromthe group consisting:

SEQ ID NOS: SEQ ID NOS:112, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, and 326; and

an Ig variable light region having an amino acid sequence selected fromthe group consisting:

SEQ ID NOS: 113, 327, 328, 329, 330, 331, 332, and 333.

According to the invention, variable heavy (VH) domains and variablelight (VL) domains of DLL4 binding proteins described herein may also beshuffled using recombinant techniques available in the art to generateand select for additional DLL4 binding proteins that comprise variouscombinations of VH and VL domains described herein.

In a preferred embodiment, a DLL4 binding protein according to theinvention binds human DLL4 (hu DLL4) and at least one other species ofDLL4. More preferably, a DLL4 binding protein described herein bindshuman DLL4 and a DLL4 selected from the group consisting of: a mouseDLL4 (mu DLL4), a cynomolgus monkey DLL4 (cynomolgus DLL4, cyno DLL4), arat DLL4, and combinations thereof.

In another embodiment, a DLL4 binding protein is a fully human antibodyor antigen binding portion thereof.

In another embodiment, a DLL4 binding protein is a CDR grafted antibody.More preferably, a DLL4 binding protein is a CDR-grafted antibody orantigen binding portion thereof comprising one or more CDRs describedabove.

Still more preferably, the CDR grafted antibody or antigen bindingportion thereof comprise a variable domain described above. Morepreferably, a CDR grafted antibody or antigen binding portion thereofcomprises two variable domains described above. Preferably, the CDRgrafted antibody or antigen binding portion thereof comprises a humanacceptor framework. More preferably, the human acceptor framework is anyone of the human acceptor frameworks described above.

More preferably, a binding protein is capable of neutralizing anactivity of a DLL4 selected from the group consisting of human DLL4,mouse DLL4, cynomolgus monkey DLL4, rat DLL4, and combinations thereof.Evaluating the neutralization of activity of DLL4 can be assessed viaseveral in vitro and in vivo assays know in the art. Exemplaryparameters for assessing neutralization of DLL4 activity include, butare not limited to, antibodies that inhibit DLL4 interaction with theNotch receptor, and/or Notch-signaling pathway with an IC₅₀ values ofabout at least 10⁻⁶ M; at least 10⁻⁷ M, or at least 10⁻⁸ M.

In one embodiment, the binding protein of the invention has an on rateconstant (K_(on)) to DLL4 of at least about 10²M⁻¹s⁻¹; at least about10³M⁻¹s⁻¹; at least about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; or atleast about 10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance.Preferably, the binding protein of the invention has an on rate constant(K_(on)) to DLL4 between 10²M⁻¹s⁻¹ to 10³M⁻¹ s⁻¹; between 10³M⁻¹s⁻¹ to10⁴M⁻¹s⁻¹; between 10⁴M⁻¹s⁻¹ to 10⁵M⁻¹s⁻¹; or between 10⁵M⁻¹s⁻¹ to10⁶M⁻¹ s⁻¹, as measured by surface plasmon resonance.

In another embodiment, the binding protein of the invention has an offrate constant (K_(off)) for DLL4 of at most about 10⁻³s⁻¹; at most about10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹; or at most about 10⁻⁶s⁻¹, as measured bysurface plasmon resonance. Preferably, the binding protein of theinvention has an off rate constant (K_(off)) to DLL4 of 10⁻³s⁻¹ to10⁻⁴s⁻¹; of 10⁻⁴s⁻¹ to 10⁻⁵s⁻¹; or of 10⁻⁵s⁻¹ to 10⁻⁶s⁻¹, as measured bysurface plasmon resonance.

In another embodiment, the binding protein of the invention has adissociation constant (K_(D)) to DLL4 of at most about 10⁻⁷ M; at mostabout 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at most about10⁻¹¹ M; at most about 10⁻¹² M; or at most 10⁻¹³ M. Preferably, thebinding protein of the invention has a dissociation constant (K_(D)) toDLL4 of 10⁻⁷ M to 10⁻⁸ M; of 10⁻⁸ M to 10⁻⁹ M; of 10⁻⁹ M to 10⁻¹⁰ M; of10⁻¹⁰ to 10⁻¹¹ M; of 10⁻¹¹ M to 10⁻¹² M; or of 10⁻¹² to M 10⁻¹³ M.

One embodiment of the invention provides an antibody constructcomprising any one of the DLL4 binding proteins disclosed above and alinker polypeptide or an immunoglobulin constant domain. In a preferredembodiment, an antibody construct according to the invention is selectedfrom the group consisting of: an immunoglobulin molecule, a monoclonalantibody, a chimeric antibody, a CDR-grafted antibody, a humanizedantibody, a Fab, a Fab′, a F(ab′)₂, a Fv, a disulfide linked Fv, a scFv,a single domain antibody, a diabody, a multispecific antibody, a dualspecific antibody, and a bispecific antibody.

In a preferred embodiment, an antibody construct of the inventioncomprises a heavy chain immunoglobulin constant domain selected from thegroup consisting of a human IgM constant domain, a human IgG1 constantdomain, a human IgG2 constant domain, a human IgG3 constant domain, ahuman IgG4 constant domain, a human IgE constant domain, and a human IgAconstant domain, and mutants of the above Ig isotypes which may alter Fcgamma receptor binding, FcRn binding, C1q binding and may alterpharmacokinetic properties and/or Fc effector functions.

In another embodiment, an antibody construct is glycosylated.Preferably, the glycosylation is a human glycosylation pattern.

In another embodiment, a DLL4 binding protein described herein isconjugated to an agent. Binding protein conjugates of the inventioninclude antibody conjugates in which an antibody construct describedherein is conjugated to an agent. Preferably, the agent is selected fromthe group consisting of: an immunoadhesion molecule, an imaging agent, atherapeutic agent, and a cytotoxic agent. In a preferred embodiment, theimaging agent is selected from the group consisting of a radiolabel, anenzyme, a fluorescent label, a luminescent label, a bioluminescentlabel, a magnetic label, and biotin. More preferably the imaging agentis a radiolabel selected from the group consisting of: ³H, ¹⁴C, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. In a preferredembodiment, the therapeutic or cytotoxic agent is selected from thegroup consisting of: an anti-metabolite, an alkylating agent, anantibiotic, a growth factor, a cytokine, an anti-angiogenic agent, ananti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.

In another embodiment, a DLL4 binding protein, an antibody construct, ora binding protein conjugate (including antibody conjugates) disclosedabove exists as a crystal. Preferably, the crystal is a carrier-freepharmaceutical controlled release crystal. In a preferred embodiment,such a crystallized binding protein, crystallized antibody construct, orcrystallized antibody conjugate has a greater half life in vivo than itssoluble counterpart. In another preferred embodiment, the crystallizedbinding protein, crystallized antibody construct, or crystallizedbinding protein conjugate (including antibody conjugate) retainsbiological activity after crystallization.

One aspect of the invention pertains to an isolated nucleic acidencoding a DLL4 binding protein, an antibody construct, a DLL4 bindingantibody conjugate, or DLL4 binding portion thereof. Particularlypreferred is an isolated nucleic acid that encodes a polypeptideselected from the group consisting of: a polypeptide comprising a heavychain variable domain, wherein the heavy chain variable domain comprisesa CDR-H1, a CDR-H2, or a CDR-H3 described above: a polypeptidecomprising a light chain variable domain, wherein the light chainvariable domain comprises a CDR-L1, a CDR-L2, or a CDR-L3 as describedabove; or a combination of both polypeptides.

A further embodiment provides a vector comprising an isolated nucleicacid disclosed above. In a preferred embodiment, the vector is selectedfrom the group consisting of: pcDNA, pTT (Durocher et al., Nucl. AcidsRes., 30(2e9): 1-9 (2002)), pTT3 (pTT with additional multiple cloningsites), pEFBOS (Mizushima et al., Nucl. Acids. Res., 18 (17): 5322(1990)), pHybE, pBV, pJV, and pBJ, and any other expression vectorssuitable for prokaryotic or eukaryotic cells.

In another aspect of the invention there is provided a host cell istransformed with the vector disclosed above. The host cell may beprokaryotic or eukaryotic cell. A preferred prokaryotic host cell isEscherichia coli. Preferably, the eukaryotic cell is selected from thegroup consisting of: a protist cell, an animal cell, a plant cell, and afungal cell. More preferably, the host cell is a mammalian cellincluding, but not limited to, CHO and COS cells. A preferred fungalcell is, but not limited to, Saccharomyces cerevisiae. A preferredinsect cell is an Sf9 cell.

Another aspect of the invention provides a method of producing a bindingprotein that binds human DLL4 comprising the step of culturing any oneof the host cells disclosed above in a culture medium under conditionssufficient to produce a binding protein that binds human DLL4. Anotherembodiment provides a binding protein produced according to the methoddisclosed above.

One embodiment provides a composition for the release of a DLL4 bindingprotein according to the invention wherein the composition comprises aformulation which in turn comprises a crystallized DLL4 binding protein,crystallized antibody construct, or crystallized binding proteinconjugate (including antibody conjugates) as disclosed above and aningredient, and further at least one polymeric carrier. Preferably, thepolymeric carrier is a polymer selected from one or more of the groupconsisting of: poly (acrylic acid), poly (cyanoacrylates), poly (aminoacids), poly (anhydrides), poly (depsipeptide), poly (esters), poly(lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly(b-hydroxybutyrate), poly (caprolactone), poly (dioxanone), poly(ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly[(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,pluronic polyols, albumin. alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polyeaccharides, blends andcopolymers thereof. Preferably the ingredient is selected from the groupconsisting of albumin, sucrose, trehalose, lactitol, gelatin,hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol andpolyethylene glycol.

Another embodiment provides a method for treating a mammal comprisingthe step of administering to the mammal an effective amount of acomposition comprising a crystallized DLL4 binding protein, acrystallized antibody construct, or a crystallized protein conjugate(including antibody conjugates) disclosed above.

The invention also provides a pharmaceutical composition comprising aDLL4 binding protein, an antibody construct, or a binding proteinconjugate (including antibody conjugates) as disclosed above and apharmaceutically acceptable carrier. In a further embodiment, thepharmaceutical composition comprises at least one additional agent. Theadditional agent may be a therapeutic agent for treating a disorder inwhich DLL4 is detrimental. Preferably, a pharmaceutical compositioncomprises an additional agent selected from the group consisting of: atherapeutic agent; an imaging agent; an antineoplastic agent; achemtherapeutic agent (such as a DNA alkylating agent, cisplatin,carboplatin, an anti-tubulin agent, paclitaxel, docetaxel, doxorubicin,gemcitabine, gemzar, an anthracycline, adriamycin, a topoisiomersase Iinhibitor, a topoisomerase II inhibitor, 5-fluorouracil (5-FU),leucovorin, irinotecan), and a receptor tyrosine kinase inhibitors(e.g., erlotinib, gefitinib), a COX-2 inhibitor (e.g., celecoxib), akinase inhibitor, and an angiogenesis inhibitor (including but notlimited to an anti-VEGF antibody or VEGF-trap); a co-stimulationmolecule blocker (including but not limited to anti-B7.1 antibody,anti-B7.2 antibody, CTLA4-Ig, anti-CD20 antibody); an adhesion moleculeblocker (including but not limited to an anti-LFA-1 antibody, ananti-E/L selectin antibody, and a small molecule inhibitor);anti-cytokine antibody or functional fragment thereof (including but notlimited to an anti-IL-18, an anti-TNF, and an anti-IL-6/cytokinereceptor antibody); methotrexate; cyclosporine; rapamycin: FK506; adetectable label or reporter molecule; a TNF antagonist; anantirheumatic; a muscle relaxant; a narcotic; a non-steroidanti-inflammatory drug (NSAID); an analgesic; an anesthetic; a sedative;a local anesthetic; a neuromuscular blocker; an antimicrobial agent; anantipsoriatic agent; a corticosteroid; an anabolic steroid; anerythropoietin; an immunization; an immunoglobulin; an immunosuppressiveagent: a growth hormone; a hormone replacement drug; aradiopharmaceutical drug; an antidepressant; an antipsychotic drug: astimulant; an asthma medication; a beta agonist; an inhaled steroid; anepinephrine or analog thereof; a cytokine; and a cytokine antagonist.

In another aspect, the invention provides a method for inhibiting humanDLL4 □activity comprising contacting human DLL4 with a binding proteindisclosed above such that human DLL4 is inhibited or neutralized. In arelated aspect, the invention provides a method for inhibiting DLL4activity in a human subject suffering from a disorder in which DLL4 isdetrimental, comprising administering to the human subject a bindingprotein disclosed above such that human DLL4 in the human subject isinhibited and treatment is achieved. Preferably, the disorder isselected from the group comprising primary and metastatic cancers,including carcinomas of breast, colon, rectum, lung, oropharynx,hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bileducts, small intestine, urinary tract (including kidney, bladder, andurothelium), female genital tract (including cervix, uterus, and ovariesas well as choriocarcinoma and gestational trophoblastic disease), malegenital tract (including prostate, seminal vesicles, testes, and germcell tumors), endocrine glands (including the thyroid, adrenal, andpituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas(including those arising from bone and soft tissues as well as Kaposi'ssarcoma), tumors of the brain, nerves, eyes, and meninges (includingastrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,neuroblastomas, Schwannomas, and meningiomas), solid tumors arising fromhematopoietic malignancies such as leukemias and lymphomas (bothHodgkin's and non-Hodgkin's lymphomas), tumor metastases, ocularneovascularization (including diabetic blindness, retinopathies,age-induced macular degeneration and rubeosis), edema, rheumatoidarthritis, multiple sclerosis, atheroscleorotic plaques, Crohn'sdisease, inflammatory bowel disease, refractory ascites, psoriasis,sarcoidosis, arterial arteriosclerosis, sepsis, peptic ulcers, burns,and pancreatitis, polycystic ovarian disease (POD), endometriosis,uterine fibroids, benign prostate hypertrophy, and other angiogenesisindependent and dependent diseases characterized by abberant DLL4activity.

In another aspect the invention provides a method of treating a patientsuffering from a disorder in which human DLL4 is detrimental comprisingthe step of administering any one of the binding proteins disclosedabove before, concurrent, or after the administration of a second agent,as discussed above. In a preferred embodiment, the second agent isselected from the group consisting of: radiotherapeutic agent; anantineoplastic agent: a chemotherapeutic agent (such as a DNA alkylatingagent, cisplatin, carboplatin, an anti-tubulin agent, paclitaxel,docetaxel, taxol, doxorubicin, gemcitabine, gemzar, an anthracycline,adriamycin, a topoisomerase I inhibitor, a topoisomerase II inhibitor,5-fluorouracil (5-FU), leucovorin, irinotecan), a receptor tyrosinekinase inhibitor (e.g., erlotinib, gefitinib), a COX-2 inhibitor (e.g.,celecoxib), a kinase inhibitor, and an angiogenesis inhibitor (includingbut not limited to an anti-VEGF antibody or VEGF-trap); a co-stimulationmolecule blocker (including but not limited to anti-B7.1, anti-B7.2,CTLA4-Ig, anti-CD20); an adhesion molecule blocker (including but notlimited to an anti-LFA-1 antibody, an anti-E/L selectin antibody, asmall molecule inhibitor); anti-cytokine antibody or functional fragmentthereof (including but not limited to anti-IL-18, anti-TNF,anti-IL-6/cytokine receptor antibodies); methotrexate: cyclosporine;rapamycin: FK506; detectable label or reporter; a TNF antagonist; anantirheumatic; a muscle relaxant; a narcotic; a non-steroidanti-inflammatory drug (NSAID); an analgesic: an anesthetic; a sedative:a local anesthetic; a neuromuscular blocker; an antimicrobial agent; anantipsoriatic drug; a corticosteroid; an anabolic steroid; anerythropoietin; an immunization; an immunoglobulin; an immunosuppressiveagent; a growth hormone; a hormone replacement drug; aradiopharmaceutical drug; an antidepressant; an antipsychotic drug; astimulant; an asthma medication; a beta agonist; an inhaled steroid: anepinephrine or analog thereof; a cytokine; and a cytokine antagonist.

In a preferred embodiment, the pharmaceutical compositions disclosedabove are administered to the subject by at least one mode selected fromthe group consisting of: parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracelebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

Another aspect of the invention provides at least one DLL4 anti-idiotypeantibody to at least one DLL4 binding protein of the present invention.The anti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one complementarily determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework region, and any portionthereof, that can be incorporated into a binding protein of the presentinvention.

Any of a variety of immunodetection assay formats may be adapted toemploy a DLL4 binding protein of the invention to detect DLL4 in amixture, solution, or biological sample. Such immunodetection assayformats include but are not limited to radioimmunoassay (RIA),immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),immunoblot (e.g, Western), immunostrips (e.g., immunodipsticks)comprising a DLL4 binding protein of the invention adsorbed orimmobilized to substrate. FACS, and the like. Detection of DLL4 using aDLL4 binding protein of the invention may be conducted in vitro on amixture, solution, or in biological sample. A biological sample that maybe contacted with binding protein of the invention to detect or measureDLL4 in the sample includes, but is not limited to, urine, saliva, oralswab (buccal, lingual, or throat swab), dermal swab, dermal scrape,rectal swab, vaginal swab, whole blood sample, plasma sample, serumsample, tissue biopsy, and any other sample obtained from an individualby a procedure known in the art. In another embodiment, a DLL4 bindingprotein may be employed to detect DLL4 in vivo such as varioustomography and scanning methods, including but not limited to X-raycomputer assisted tomography (CT), magnetic resonance imaging (MRI), andpositron emission tomography (PET).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to DLL4 binding proteins, particularly anti-DLL4antibodies, or antigen-binding portions thereof that bind DLL4. Variousaspects of the invention relate to antibodies and antibody fragments,and pharmaceutical compositions thereof, as well as nucleic acids,recombinant expression vectors, and host cells for making suchantibodies and fragments. Methods of using the antibodies of theinvention to detect human DLL4 or murine DLL4, methods to inhibit humanor mouse DLL4 and/or human or mouse VEGFR2 or VEGR1 activity, either invitro or in vivo, and methods to regulate gene expression are alsoencompassed by the invention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including.” as well as other forms, such as “includes” and “included,”is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric. Use of “polypeptide” hereinis intended to encompass polypeptide and fragments and variants(including fragments of variants) thereof, unless otherwise stated. Foran antigenic polypeptide, a fragment of polypeptide optionally containsat least one contiguous or nonlinear epitope of polypeptide. The preciseboundaries of the at least one epitope fragment can be confirmed usingordinary skill in the art. The fragment comprises at least about 5contiguous amino acids, such as at least about 10 contiguous aminoacids, at least about 15 contiguous amino acids, or at least about 20contiguous amino acids. A variant of polypeptide is as described herein.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

The term “human DLL4” (abbreviated herein as “hDLL4” or “huDLL4”), asused herein, includes several EGF-like domains and a DSL domain that isrequired for receptor binding. The term includes a protein comprisingabout 74-75 kDa. The structure and deduced DNA and protein sequences ofhuman DLL4 is described further in, for example. Shutter et al., Genes &Dev., 4: 1313-1318 (2000). The term “human DLL4” is intended to includerecombinant human DLL4 (rh DLL4), which can be prepared by standardrecombinant expression methods.

“Biological activity”, as used herein with respect to DLL4, refers toall inherent biological properties of DLL4. Biological properties ofDLL4 include, but are not limited to, binding a Notch receptor,activating a Notch receptor, negatively regulating VEGF signaling,repressing VEGFR2, and inducing VEGR1.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, means that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species: for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Nonlimitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementarydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4. IgA1and IgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of anantibody mediates several important effector functions, e.g., cytokineinduction. ADCC, phagocytosis, complement dependent cytotoxicity (CDC),and half-life: clearance rate of antibody and antigen-antibodycomplexes. In some cases these effector functions are desirable for atherapeutic antibody but in other cases might be unnecessary or evendeleterious, depending on the therapeutic objectives. Certain human IgGisotypes, particularly IgG1 and IgG3, mediate ADCC and CDC via bindingto FcγRs and complement C1q, respectively. Neonatal Fc receptors (FcRn)are the critical components determining the circulating half-life ofantibodies. In still another embodiment, at least one amino acid residueis replaced in the constant region of the antibody, for example, the Fcregion of the antibody, such that effector functions of the antibody arealtered. The dimerization of two identical heavy chains of animmunoglobulin is mediated by the dimerization of CH3 domains and isstabilized by the disulfide bonds within the hinge region (Huber et al.,Nature, 264: 415-420 (1976); Thies et al., J. Mol. Biol., 293: 67-79(1999)). Mutation of cysteine residues within the hinge regions toprevent heavy chain-heavy chain disulfide bonds will destabilizedimerization of CH3 domains. Residues responsible for CH3 dimerizationhave been identified (Dall'Acqua, Biochem., 37: 9266-9273 (1998)).Therefore, it is possible to generate a monovalent half-Ig.Interestingly, these monovalent half Ig molecules have been found innature for both IgG and IgA subclasses (Seligman, Ann. Immunol., 129:855-70 (1978): Biewenga et al., Clin. Exp. Immunol., 51: 395-400(1983)). The stoichiometry of FcRn: Ig Fc region has been determined tobe 2:1 (West et al., Biochem., 39: 9698-9708 (2000)), and half Fc issufficient for mediating FcRn binding (Kim et al., Eur. J. Immunol., 24:542-548 (1994)). Mutations to disrupt the dimerization of CH3 domain maynot have greater adverse effect on its FcRn binding as the residuesimportant for CH3 dimerization are located on the inner interface of CH3b sheet structure, whereas the region responsible for FcRn binding islocated on the outside interface of CH2-CH3 domains. However, the halfIg molecule may have certain advantages in tissue penetration due to itssmaller size in comparison to that of a regular antibody. In oneembodiment, at least one amino acid residue is replaced in the constantregion of a binding protein of the invention, for example the Fc region,such that the dimerization of the heavy chains is disrupted, resultingin half Ig molecules. The anti-inflammatory activity of IgG iscompletely dependent on sialylation of the N-linked glycan of the IgG Fcfragment. The precise glycan requirements for anti-inflammatory activityhas been determined, such that an appropriate IgG1 Fc fragment can becreated, thereby generating a fully recombinant, sialylated IgG1 Fc withgreatly enhanced potency (Anthony et al., Science, 320: 373-376 (2008)).

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retains the ability to bind specifically to an antigen(i.e., to a particular epitope of an antigen, such as an epitope ofDLL4). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormultispecific formats; specifically binding to two or more differentantigens (or two or more different epitopes of the same antigen).Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL. VH, CL, and CH1 domains: (ii)a F(ab)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region: (iii) an Fd fragmentconsisting of the VH and CH1 domains: (iv) an Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody; (v) a dAb fragment(Ward et al., Nature, 341:544-546 (1989); PCT Publication No. WO90/05144 A1), which comprises a single variable domain; and (vi) anisolated complementary determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment. VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv): see, e.g., Bird et al., Science, 242: 423-426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding portion” of an antibody. Other forms of singlechain antibodies, such as diabodies, are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see, e.g., Holliger et al.,Proc. Natl. Acad Sci. USA, 90: 6444-(448 (1993); Poljak. R. J.,Structure, 2: 1121-1123 (1994)). Such antibody binding portions areknown in the art (see, Kontermann and Dubel eds., Antibody Engineering(Springer-Verlag. New York, 2001), p. 790 (ISBN 3-540-41354-5)). Inaddition, single chain antibodies also include “linear antibodies”comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al. Protein Eng., 8(10): 1057-1062 (1995);and U.S. Pat. No. 5,641,870).

The term “antibody construct” (or “DLL4 antibody construct”) as usedherein refers to a polypeptide comprising one or more the antigenbinding portions of the invention linked to a linker polypeptide or animmunoglobulin constant domain. Linker polypeptides comprise two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Such linker polypeptides are well knownin the art (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). Animmunoglobulin constant domain refers to a heavy or light chain constantdomain. Human IgG heavy chain and light chain constant domain amino acidsequences are known in the art and represented in Table 1.

TABLE 1Sequence of human IgG heavy chain constant domain and light chainconstant domain Sequence Sequence Protein Identifier12345678901234567890123456789012 Ig gamma-1 constant SEQ IDASTKGPSVFFLAPSSKSTSGGTAALGCLVKDY region NO: 2FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig gamma-1 constant SEQ IDASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY region mutant NO: 3FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig Kappa constant SEQ IDTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY region NO: 4PREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda constant SEQ ID QPKAAPSVTLFPPSSEELQANKATLVCLISDFregion NO: 5 YPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al., Human Antibodies andHybridomas, 6: 93-101 (1995)) and use of a cysteine residue, a markerpeptide, and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov et al., Mol. Immunol., 31:1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)2 fragments,can be prepared from whole antibodies using conventional techniques,such as papain or pepsin digestion, respectively, of whole antibodies.Moreover, antibodies, antibody portions, and immunoadhesion moleculescan be obtained using standard recombinant DNA techniques, as describedherein and known in the art.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hDLL4 is substantially free of antibodies that specifically bindantigens other than hDLL4). An isolated antibody that specifically bindshDLL4 may, however, have cross-reactivity to other antigens, such asDLL4 molecules from other species (e.g., muDLL4). Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals.

The term “monoclonal antibody” and abbreviations “MAb” and “mAb”, asused herein, refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a single antigen.Furthermore, in contrast to polyclonal antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each mAb is directed against a singledeterminant on the antigen. The modifier “monoclonal” is not to beconstrued as requiring production of the antibody by any particularmethod.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created, orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library(Hoogenboom, Trends Biotechnol., 15:62-70 (1997); Azzazy and Highsmith,Clin. Biochem., 35: 425-445 (2002); Gavilondo and Larrick,BioTechniques, 29: 128-145 (2000); Hoogenboom and Chames, Immunol.Today, 21: 371-378 (2000)), antibodies isolated from an animal (e.g., amouse) that is transgenic for human immunoglobulin genes (see, Taylor etal., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermann and Green. Curr.Opin. Biotechnol., 13: 593-597 (2002): Little et al., Immunol. Today,21: 364-370 (2000)) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

As used herein, the term “CDR” refers to a complementary determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated “CDR1”, “CDR2”, and “CDR3”, for each of the variableregions. The term “CDR set” as used herein refers to a group of threeCDRs that occur in a single variable region that binds the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda. Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as “Kabat CDRs”. Chothiaand coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987).Chothia et al., Nature, 342: 877-883 (1989)) found that certainsub-portions within Kabat CDRs adopt nearly identical peptide backboneconformations, despite having great diversity at the level of amino acidsequence. These sub-portions were designated as “L1”, “L2”, and “L3”, or“H1”, “H2”, and “H3”, where the “L” and the “H” designate the lightchain and the heavy chain regions, respectively. These regions may bereferred to as “Chothia CDRs”, which have boundaries that overlap withKabat CDRs. Other boundaries defining CDRs overlapping with the KabatCDRs have been described by Padlan, FASEB J., 9: 133-139 (1995) andMacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDRboundary definitions may not strictly follow one of the herein systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, although certainembodiments use Kabat or Chothia defined CDRs.

The terms “Kabat numbering,” “Kabat definitions”, and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al., Ann. NY Acad. Sci., 190: 382-391(1971) and Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 (1991)). For the heavy chain variable region(VH), the hypervariable region ranges from amino acid positions 31 to 35for CDR1, amino acid positions 50 to 65 for CDR2, and amino acidpositions 95 to 102 for CDR3. For the light chain variable region (VL),the hypervariable region ranges from amino acid positions 24 to 34 forCDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions89 to 97 for CDR3.

The growth and analysis of extensive public databases of amino acidsequences of variable heavy and light regions over the past twenty yearshave led to the understanding of the typical boundaries betweenframework regions (FR) and CDR sequences within variable regionsequences and enabled persons skilled in this art to accuratelydetermine the CDRs according to Kabat numbering. Chothia numbering, orother systems. See, e.g., Martin, “Protein Sequence and StructureAnalysis of Antibody Variable Domains,” In Kontermann and Dübel, eds.,Antibody Engineering (Springer-Verlag, Berlin, 2001), chapter 31, pages432-433. A useful method of determining the amino acid sequences ofKabat CDRs within the amino acid sequences of variable heavy (VH) andvariable light (VL) regions is provided below:

To identify a CDR-L1 amino acid sequence:

-   -   Starts approximately 24 amino acid residues from the amino        terminus of the VL region;    -   Residue before the CDR-L1 sequence is always cysteine (C);    -   Residue after the CDR-L1 sequence is always tryptophan (W)        residue, typically Trp-Tyr-Gln (W-Y-Q), but also Trp-Leu-Gln        (W-L-Q), Trp-Phe-Gln (W-F-Q), and Trp-Tyr-Leu (W-Y-L);    -   Length is typically 10 to 17 amino acid residues.

To identify a CDR-L2 amino acid sequence:

-   -   Starts always 16 residues after the end of CDR-L1;    -   Residues before the CDR-L2 sequence are generally Ile-Tyr (1-Y),        but also Val-Tyr (V-Y), Ile-Lys (I-K), and Ile-Phe (I-F);    -   Length is always 7 amino acid residues.

To identify a CDR-L3 amino acid sequence:

-   -   Starts always 33 amino acids after the end of CDR-L2;    -   Residue before the CDR-L3 amino acid sequence is always a        cysteine (C);    -   Residues after are always Phe-Gly-X-Gly (F-G-X-G) (SEQ ID        NO:374), where X is any amino acid;    -   Length is typically 7 to 11 amino acid residues.

To identify a CDR-H1 amino acid sequence:

-   -   Starts approximately 31 amino acid residues from amino terminus        of VH region and always 9 residues after a cysteine (C);    -   Residues before are always Cys-X-X-X-X-X-X-X-X (SEQ ID NO:375),        where X is any amino acid;    -   Residue after is always a Trp (W), typically Trp-Val (W-V), but        also Trp-Ile (W-I), and Trp-Ala (W-A);    -   Length is typically 5 to 7 amino acid residues.

To identify a CDR-H2 amino acid sequence:

-   -   Starts always 15 amino acid residues after the end of CDR-H1;    -   Residues before are typically Leu-Glu-Trp-Ile-Gly (L-E-W-I-G)        (SEQ ID NO:376), but other variations also;    -   Residues after are        Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala        (K/R-L/I/V/F/T/A-T/S/l/A);    -   Length is typically 16 to 19 amino acid residues.

To identify a CDR-H3 amino acid sequence:

-   -   Starts always 33 amino acid residues after the end of CDR-H2 and        always 3 after a cysteine (C)′    -   Residues before are always Cys-X-X (C-X-X), where X is any amino        acid, typically Cys-Ala-Arg (C-A-R);    -   Residues after are always Trp-Gly-X-Gly (W-G-X-G) (SEQ ID        NO:377), where X is any amino acid;    -   Length is typically 3 to 25 amino acid residues.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like,” i.e., more similar tohuman germline variable sequences. A “humanized antibody” is an antibodyor a variant, derivative, analog, or fragment thereof, whichimmunospecifically binds to an antigen of interest and which comprises aframework (FR) region having substantially the amino acid sequence of ahuman antibody and a complementary determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody. As usedherein, the term “substantially” in the context of a CDR refers to a CDRhaving an amino acid sequence at least 80%, at least 85%, at least 90%,at least 95%, at least 98% or at least 99% identical to the amino acidsequence of a non-human antibody CDR. A humanized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a human immunoglobulin consensus sequence. In anembodiment, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

A humanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA, and IgE, and any isotype, includingwithout limitation IgG1, IgG2, IgG3, and IgG4. A humanized antibody maycomprise sequences from more than one class or isotype, and particularconstant domains may be selected to optimize desired effector functionsusing techniques well known in the art.

The framework regions and CDRs of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion, and/or deletion of at least one amino acid residue so thatthe CDR or framework residue at that site does not correspond to eitherthe donor antibody or the consensus framework. In a preferredembodiment, such mutations, however, will not be extensive. Usually, atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See, e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, 1987)). A “consensusimmunoglobulin sequence” may thus comprise a “consensus frameworkregion(s)” and/or a “consensus CDR(s)”. In a family of immunoglobulins,each position in the consensus sequence is occupied by the amino acidoccurring most frequently at that position in the family. If two aminoacids occur equally frequently, either can be included in the consensussequence.

An “affinity matured” antibody is an antibody with one or morealterations in one or more CDRs thereof which result in an improvementin the affinity of the antibody for a target antigen, compared to aparent antibody which does not possess the alteration(s). Exemplaryaffinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. A variety of procedures for producingaffinity matured antibodies are known in the art. For example, Marks etal., BioTechnology, 10: 779-783 (1992) describes affinity maturation byVH and VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad Sci. USA, 91:3809-3813 (1994): Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

The term “multivalent binding protein” denotes a binding proteincomprising two or more antigen binding sites (also referred to herein as“antigen binding domains”). A multivalent binding protein is preferablyengineered to have three or more antigen binding sites, and is generallynot a naturally occurring antibody. The term “multispecific bindingprotein” refers to a binding protein capable of binding two or morerelated or unrelated targets, including a binding protein capable ofbinding two or more different epitopes of the same target molecule.

The term “bispecific antibody”, as used herein, refers to full-lengthantibodies that are generated by quadroma technology (see Milstein etal., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of twodifferent monoclonal antibodies (see. Staerz et al., Nature, 314(6012):628-631 (1985)), or by knob-into-hole or similar approaches whichintroduces mutations in the Fc region (see Holliger et al., Proc. Natl.Acad Sci. USA, 90(14): 6444-6448 (1993)), resulting in multipledifferent immunoglobulin species of which only one is the functionalbispecific antibody. By molecular function, a bispecific antibody bindsone antigen (or epitope) on one of its two binding arms (one pair ofHC/LC), and binds a different antigen (or epitope) on its second arm (adifferent pair of HC/LC). By this definition, a bispecific antibody hastwo distinct antigen binding arms (in both specificity and CDRsequences), and is monovalent for each antigen it binds to.

The term “dual-specific antibody”, as used herein, refers to full-lengthantibodies that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT publication WO02/02773). Accordingly a dual-specific binding protein has two identicalantigen binding arms, with identical specificity and identical CDRsequences, and is bivalent for each antigen to which it binds.

“Dual variable domain” (“DVD”) binding proteins of the inventioncomprise two or more antigen binding sites and may be divalent (twoantigen binding sites), tetravalent (four antigen binding sites), ormultivalent binding proteins. DVDs may be monospecific, i.e., capable ofbinding one antigen (or one specific epitope), or multispecific, i.e.,capable of binding two or more antigens (i.e., two or more epitopes ofthe same target antigen molecule or two or more epitopes of differenttarget antigens). A preferred DVD binding protein comprises two heavychain DVD polypeptides and two light chain DVD polypeptides is referredto as a “DVD immunoglobulin” or “DVD-Ig”. Such a DVD-Ig binding proteinis thus tetrameric and reminiscent of an IgG molecule, but provides moreantigen binding site than an IgG molecule. Thus, each half of atetrameric DVD-Ig molecule is reminiscent of one half of an IgG moleculeand comprises a heavy chain DVD polypeptide and a light chain DVDpolypeptide, but unlike a pair of heavy and light chains of an IgGmolecule that provide a single antigen bindind domain, a pair of heavyand light chains of a DVD-Ig provide two or more antigen binding sites.

Each antigen binding site of a DVD-Ig binding protein is derived from adonor (“parental”) monoclonal antibody and thus comprises a heavy chainvariable domain (VH) and a light chain variable domain (VL) with a totalof six CDRs involved in antigen binding per antigen binding site.Accordingly, a DVD-Ig binding protein that binds two different epitopes(i.e., two different epitopes of two different antigen molecules or twodifferent epitopes of the same antigen molecule) comprises an antigenbinding site derived from a first parental monoclonal antibody and anantigen binding site of a second parental monoclonal antibody.

A description of the design, expression, and characterization of DVD-Igbinding molecules is provided in PCT Publication No. WO 2007/024715,U.S. Pat. No. 7,612,181, and Wu et al., Nature Biotech., 25: 1290-1297(2007). A preferred example of such DVD-Ig molecules comprises a heavychain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first heavy chain variable domain, VD2 is a secondheavy chain variable domain, C is a heavy chain constant domain. X1 is alinker with the proviso that it is not CH1, X2 is an Fc region, and n is0 or 1, but preferably 1; and a light chain that comprises thestructural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first lightchain variable domain, VD2 is a second light chain variable domain. C isa light chain constant domain. X is a linker with the proviso that it isnot CH1, and X2 does not comprise an Fc region; and n is 0 or 1, butpreferably 1. Such a DVD-Ig may comprise two such heavy chains and twosuch light chains, wherein each chain comprises variable domains linkedin tandem without an intervening constant region between variableregions, wherein a heavy chain and a light chain associate to formtandem functional antigen binding sites, and a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with four functional antigen binding sites.In another example, a DVD-Ig molecule may comprise heavy and lightchains that each comprise three variable domains (VD1. VD2. VD3) linkedin tandem without an intervening constant region between variabledomains, wherein a pair of heavy and light chains may associate to formthree antigen binding sites, and wherein a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with six antigen binding sites.

In a preferred embodiment, a DVD-Ig binding protein according to theinvention not only binds the same target molecules bound by its parentalmonoclonal antibodies, but also possesses one or more desirableproperties of one or more of its parental monoclonal antibodies.Preferably, such an additional property is an antibody parameter of oneor more of the parental monoclonal antibodies. Antibody parameters thatmay be contributed to a DVD-Ig binding protein from one or more of itsparental monoclonal antibodies include, but are not limited to, antigenspecificity, antigen affinity, potency, biological function, epitoperecognition, protein stability, protein solubility, productionefficiency, immunogenicity, pharmacokinetics, bioavailability, tissuecross reactivity, and orthologous antigen binding.

A DVD-Ig binding protein according to the invention binds at least oneepitope of a human DLL4 protein. Non-limiting examples of a DVD-Igbinding protein according to the invention include a DVD-Ig bindingprotein that binds one or more epitopes of human DLL4, a DVD-Ig bindingprotein that binds an epitope of a human DLL4 and an epitope of a DLL4of another species (for example, mouse), and a DVD-Ig binding proteinthat binds an epitope of a human DLL4 and an epitope of another targetmolecule (for example, VEGFR2 or VEGFR1).

A “functional antigen binding site” of a binding protein is one that iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

As used herein, the terms “acceptor” and “acceptor antibody” refer to anantibody or nucleic acid sequence providing or encoding at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions (FRs). Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol., 196: 901-917 (1987): Chothiaet al., J. Mol. Biol., 227: 799-817 (1992), both are incorporated hereinby reference). According to Chothia et al., critical portions of theCDRs of many antibodies have nearly identical peptide backboneconfirmations despite great diversity at the level of amino acidsequence. Each canonical structure specifies primarily a set of peptidebackbone torsion angles for a contiguous segment of amino acid residuesforming a loop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems (for example, see above), the meaning of a framework sequence issubject to correspondingly different interpretations. The six CDRs(CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavychain) also divide the framework regions on the light chain and theheavy chain into four sub-regions (FR1, FR2, FR3, and FR4) on eachchain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2and FR3, and CDR3 between FR3 and FR4. Without specifying the particularsub-regions as FR1. FR2. FR3, or FR4, a framework region, as referred byothers, represents the combined FR's within the variable region of asingle, naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention the human heavy chain and lightchain acceptor sequences are selected from the sequences described inTable 2 and Table 3.

TABLE 2 Heavy Chain Acceptor Sequences Protein region/ SEQ ID ClosestSequence NO.: Germline Family 12345678901234567890123456789012  6VH2-70/JH6 FR1 EVTLRESGPALVKPTQTLTLTCTFSGFSLS  7 VH2-70/JH6 FR2WIRQPPGKALEWLA  8 VH2-70/JH6 FR3 RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR  9VH2-70/JH6 FR4 WGQGTTVTVSS 10 VH2-26/JH6 FR1EVTLKESGPVLVKPTETLTLTCTVSGFSLS  7 VH2-26/JH6 FR2 WIRQPPGKALEWLA 11VH2-26/JH6 FR3 RLTISKDTSKSQVVLTMTNMDPVDTATYYCAR  9 VH2-26/JH6 FR4WGQGTTVTVSS 12 VH3-72/JH6 FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 13VH3-72/JH6 FR2 WVRQAPGKGLEWVG 14 VH3-72/JH6 FR3RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR  9 VH3-72/JH6 FR4 WGQGTTVTVSS 15VH3-21/JH6 FR1 EVQLVESGGGLVKPGGSLRLSCAASGFTFS 16 VH3-21/JH6 FR2WVRQAPGKGLEWVS 17 VH3-21/JH6 FR3 RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR  9VH3-21/JH6 FR4 WEQGTTVTVSS 18 VH1-69/JH6 FR1EVQLVQSGAEVKKPGSSVKVSCKASGGTFS 19 VH1-69/JH6 FR2 WVRQAPGQGLEWMG 20VH1-69/JH6 FR3 RVTITADKSTSTAYMELSSLRSEDTAVYYCAR  9 VH1-69/JH6 FR4WGQGTTVTVSS 21 VH1-18/JH6 FR1 EVQLVQSGAEVKKPGASVKVSCKASGYTFT 19VH1-18/JH6 FR2 WVRQAPGQGLEWMG 22 VH1-18/JH6 FR3RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR  9 VH1-18/JH6 FR4 WGQGTTVTVSS 35IGHV4-59 FR1 EVQLQESGPGLVKPSETLSLTCTVSGGSIS 36 IGHV4-59 FR2WIRQPPGKGLEWIG 37 IGHV4-59 FR3 RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 38IGHV4-59/JH FR4 WGQGTLVTVSS 39 IGHV3-66 FW 1EVQLVESGGGLVQPGGSLRLSCAVSGGSIS 40 IGHV3-66 FW2 WIRQAPGKGLEWIG 41IGHV3-66 FW3 RVTISVDTSKNSFYLQMNSLRAEDTAVYYCAR 42 IGHV3-66/JH FW4WGQGTLVTVSS 43 IGHV4-59 FR1 EVQLQESGPGLVKPGETLSLTCTVSGGSIS 44IGHV4-59 FR2 WIRQAPGKGLEWIG 45 IGHV4-59 FR3RVTISVDTSKNQFYLKLSSVRAEDTAVYYCAR 46 IGHV4-59/JH FR4 WGQGTLVTVSS 47IGHV5-51 FR1 EVQLVQSGTEVKKPGESLKISCKVSGGSIS 48 IGHV5-51 FR2WIRQMPGKGLEWIG 49 IGHV5-51 FR3 QVTISVDTSFNTFFLQWSSLKASDTAMYYCAR 50IGHV5-51/JH FR4 WGQGTMVTVSS 51 IGHV2-70 FR1EVTLRESGPALVKPTQTLTLTCTVSGGSIS 52 IGHV2-70 FR2 WIRQPPGKGLEWIG 53IGHV2-70 FR3 RVTISVDTSKNQFVLTMTNMDPVDTATYYCAR 54 IGHV2-70/JH FR4WGQGTTVTVSS 55 IGHV3-15 FR1 EVQLLESGGGLVKSGGSLRLSCAASGFTFR 56IGHV3-15 FR2 WVRQAPGKGLEWVA 57 IGHV3-15 FR3RFTISRDNSKNTLYLQLNSLRAEDTAVYYCAK 58 IGHV3-15/JH FR4 WGQGTMVTVSS 59IGHV3-43 FR1 EVQLVESGGGVVQPGGSLRLSCAASGFTFG 60 IGHV3-43 FR2WVRQAPGKGLEWVA 61 IGHV3-43 FR3 RFTISRDNSKNTLYLOLNSLRAEDTAVYYCAK 62IGHV3-43/JH FR4 WGQGTMVTVSS

TABLE 3 Light Chain Acceptor Sequences Protein region/ SEQ ID ClosestSequence NO.: Germline Family 12345678901234567890123456789012 23B3/JK4 FR1 DIVMTQSPDSLAVSLGERATINC 24 B3/JK4 FR2 WYQQKPGQPPKLLIY 25B3/JK4 FR3 GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 26 B3/JK4 FR4 FGGGTKVEIKR 27L2/JK4 FR1 EIVMTQSPATLSVSPGERATLSC 28 L2/JK4 FR2 WYQQKPGQAPRLLIY 29L2/JK4 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC 26 L2/JK4 FR4 FGGGTKVEIKR 30L15/JK4 FR1 DIQMTQSPSSLSASVGDRVTITC 31 L15/JK4 FR2 WYQQKPEKAPKSLIY 32L15/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 26 L15/JK4 FR4 FGGGTKVEIKR33 L5/JK4 FR1 DIQMTQSPSSVSASVGDRVTITC 34 L5/JK4 FR2 WYQQKPGKAPKLLIY 32L5/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 26 L5/JK4 FR4 FGGGTKVEIKR 63IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 64 IGLV3-1 FR2 WYQQKPGQSPVLVIY 65IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADYYC 66 IGLV3-1 JL FR4FGYGTKVTVL 67 IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 68 IGLV3-1 FR2WYQQKPGQSPVLVIY 69 IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADYYC 70IGLV3-1/JL FR4 GGGTKLTVLG 71 IGLV3-1 FR1 YELTQPPSVSVSPGQTASITC 72IGLV3-1 FR2 WYQQKPGQSPVLVIY 73 IGLV3-1 FR3GIPERFSGSNSGDTATLTISGTQPMDEADYYC 74 IGLV3-1/JL FR4 GGGTKLTVLG 75IGLV3-1 FR1 LYVLTQPPSVSVSPGQTASITC 76 IGLV3-1 FR2 WYQQKPGQSPVLVIY 77IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQTMDEADYLC 78 IGLV3-1 JL FR4FGGGTKVTVLG 79 IGKV6D-21 FR1 EYVLTQSPDFQSVTPKEKVTITC 80 IGKV6D-21 FR2WYQQKPDQSPKLVIY 81 IGKV6D-21 FR3 GVPSRFSGSNSGDDATLTINSLEAEDAATYYC 82IGKV6D-21/JK FR4 FGQGTKVEIKR 83 IGKV3D-15 FR1 EYVLTQSPATLSVSPGERATLSC 84IGKV3D-15 FR2 WYQQKPGQSPRLVIY 85 IGKV3D-15 FR3DIPARFSGSNSGDEATLTISSLQSEDFAVYYC 86 IGKV3D-15/JK FR4 FGQGTRLEIKR 87IGKV4-1 FRl DYVLTQSPDSLAVSLGERATINC 88 IGKV4-1 FR2 WYQQKPGQSPKLVIY 89IGKV4-1 FR3 GIFDRFSGSNSGDDATLTISSLQAEDVAVYYC 90 IGKV4-1/JK FR4FGGGTKVEIKR 91 IGLV3-1 FR1 LPVLTQPPSVSVSPGQTASITC 92 IGLV3-1 FR2WYQQKPGQSPVLVIY 93 IGLV3-1 FR3 GIPERFSGSNSGNTATLTISGTQTMDEADYLC 94IGLV3-1/JL FR4 FGGGTKVTVL 95 IGLV3-1 FRl SYELTQPPSVSVSPGQTASITC 96IGLV3-1 FR2 WYQQKPGQSPVLVIY 97 IGLV3-1 FR3GIPERFSGSNSGNTATLTISGTQTMDEADYLC 98 IGLV3-1 JL FR4 FGGGTKLTVL

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol., 22(3):183-200 (2002); Marchalonis et al., Adv. Exp. Med. Biol., 484:13-30(2001)). One of the advantages provided by various embodiments of thepresent invention stems from the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

As used herein, the term “key residue” refers to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (J. Mol. Biol., 224: 487-499 (1992)).Vernier zone residues form a layer underlying the CDRs and may impact onthe structure of CDRs and the affinity of the antibody.

As used herein, the term “neutralizing” refers to counteracting thebiological activity of an antigen when a binding protein specificallybinds the antigen. In an embodiment, the neutralizing binding proteinbinds an antigen reduces its biologically activity by at least about20%, 40%, 60%, 80%, 85%, or more.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-hDLL4 antibody that binds to an DLL4 antigen and/or theneutralizing potency of an antibody, or an anti-hDLL4 antibody whosebinding to hDLL4 inhibits the biological activity of hDLL4, e.g.inhibition of receptor binding in a ligand-receptor binding assay orinhibition of receptor activation in a human Notch reporter assay, orstimulation of endothelial cell proliferation in a endothelial cellsprouting assay.

The term “epitope” includes any polypeptide determinant thatspecifically binds to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. An epitope thus consists of the amino acid residues of aregion of an antigen (or fragment thereof) known to bind to thecomplementary site on the specific binding partner. An antigen orantigenic fragment can contain more than one epitope. Thus, it isunderstood by persons skilled in this art that every “antigen bindingsite” of an antibody molecule binds an epitope of an antigen moleculeand every antigen molecule may have one, two, several, or many epitopes.Moreover, it is understood by persons skilled in this art that twoindependently isolated antibodies to an antigen molecule may bind at thesame epitope or at two different epitopes on the antigen molecule.

In certain embodiments, an antibody is said to specifically bind anantigen when it recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. Antibodies are said to “bind to the sameepitope” if the antibodies cross-compete (one prevents the binding ormodulating effect of the other). In addition, structural definitions ofepitopes (overlapping, similar, identical) are informative, butfunctional definitions are often more relevant as they encompassstructural (binding) and functional (modulation, competition)parameters.

The term “surface plasmon resonance,” as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore® system(BIAcore International AB, a GE Healthcare company, Uppsala, Sweden andPiscataway, N.J., US). For further descriptions, see Jönsson et al.,Ann. Biol. Clin., 51: 19-26 (1993); Jönsson et al. BioTechniques, 11:620-627 (1991); Jönsson et al., J. Mol. Recognit., 8: 125-131 (1995);and Johnsson et al., Anal. Biochem., 198: 268-277 (1991).

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of a binding protein (e.g., an antibody) to acognate partner (e.g., an antigen) to form a binding partner/cognatepartner (e.g., antibody/antigen) complex as is known in the art. The“K_(on)” also is known by the terms “association rate constant,” or“k_(a),” as used interchangeably herein. This value indicating thebinding rate of an antibody to its target antigen or the rate of complexformation between an antibody and antigen also is shown by the equation:

Antibody (“Ab”)+Antigen (“Ag”)→Ab-Ag.

The term “K_(off),” as used herein, is intended to refer to the off rateconstant for dissociation of a binding protein (e.g., an antibody) fromthe, e.g., antibody/antigen complex as is known in the art. The“K_(off)” also is known by the terms “dissociation rate constant” or“k_(d)” as used interchangeably herein. This value indicates thedissociation rate of an antibody from its target antigen or separationof Ab-Ag complex over time into free antibody and antigen as shown bythe equation below:

Ab+Ag←Ab-Ag.

The terms “equilibrium dissociation constant” or “K_(D)”, as usedinterchangeably herein, refer to the value obtained in a titrationmeasurement at equilibrium, or by dividing the dissociation rateconstant (K_(off)) by the association rate constant (K_(on)). Theassociation rate constant, the dissociation rate constant, and theequilibrium dissociation constant are used to represent the bindingaffinity of an antibody to an antigen. Methods for determiningassociation and dissociation rate constants are well known in the art.Using fluorescence-based techniques offers high sensitivity and theability to examine samples in physiological buffers at equilibrium.Other experimental approaches and instruments such as a BIAcore® surfaceplasmon resonance (biomolecular interaction analysis) assay can be used(e.g., instrument available from BIAcore International AB, a GEHealthcare company, Uppsala, Sweden). Additionally, a KinExA® (KineticExclusion Assay) assay, available from Sapidyne Instruments (Boise,Idaho) can also be used.

“Label” and “detectable label” mean a moiety attached to a specificbinding partner, such as an antibody or an analyte bound by theantibody, e.g., to render the reaction between members of a specificbinding pair, such as an antibody and an analyte, detectable. Thespecific binding partner, e.g., antibody or analyte, so labeled isreferred to as “detectably labeled”. Thus, the term “labeled bindingprotein” as used herein, refers to a protein with a label incorporatedthat provides for the identification of the binding protein. In anembodiment, the label is a detectable marker that can produce a signalthat is detectable by visual or instrumental means, e.g., incorporationof a radiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by a marked avidin (e.g., an avidin or astreptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm); chromogens, fluorescent labels(e.g., FITC, rhodamine, and lanthanide phosphors), enzymatic labels(e.g., horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, and epitope tags); and magnetic agents, such as gadoliniumchelates. Representative examples of labels commonly employed forimmunoassays include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein.Other labels are known in the art or described herein. In this regard,the moiety itself may not be detectably labeled but may becomedetectable upon reaction with yet another moiety. Use of “detectablylabeled” is intended to encompass the latter type of detectablelabeling.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably, the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof.

The terms “crystal” and “crystallized” as used herein, refer to abinding protein (e.g., an antibody), or antigen binding portion thereof,that exists in the form of a crystal. Crystals are one form of the solidstate of matter, which is distinct from other forms such as theamorphous solid state or the liquid crystalline state. Crystals arecomposed of regular, repeating, three-dimensional arrays of atoms, ions,molecules (e.g., proteins such as antibodies), or molecular assemblies(e.g., antigen/antibody complexes, including Fab/antigen complexes).These three-dimensional arrays are arranged according to specificmathematical relationships that are well-understood in the field. Thefundamental unit, or building block, that is repeated in a crystal iscalled the asymmetric unit. Repetition of the asymmetric unit in anarrangement that conforms to a given, well-defined crystallographicsymmetry provides the “unit cell” of the crystal. Repetition of the unitcell by regular translations in all three dimensions provides thecrystal. See, Giegé et al., In Crystallization of Nucleic Acids andProteins, a Practical Aporoach. 2nd ed., (Ducruix and Giegé, eds.)(Oxford University Press, New York, 1999), chapter 1, pages 1-16.

The term “polynucleotide” means a polymeric form of two or morenucleotides, either ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

The term “isolated polynucleotide” shall mean a polynucleotide (e.g., ofgenomic, cDNA, or synthetic origin, or some combination thereof) that,by virtue of its origin, the “isolated polynucleotide” is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “vector,” is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions. RNA versions of vectors(including RNA viral vectors) may also find use in the invention.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include expression controlsequences that are contiguous with a gene of interest, expressioncontrol sequences that act in trans, i.e., located on a differentnucleic acid molecule than a gene of interest, as well as expressioncontrol sequences that are located on the same nucleic acid molecule as,but at a distance from, a gene of interest. The term “expression controlsequence” as used herein refers to polynucleotide sequences which arenecessary to effect the expression and processing of coding sequences towhich they are ligated. Expression control sequences include appropriatetranscription initiation, termination, promoter and enhancer sequences:efficient RNA processing signals such as splicing and polyadenylationsignals, sequences that stabilize cytoplasmic mRNA; sequences thatenhance translation efficiency (i.e., Kozak consensus sequence);sequences that enhance protein stability; and when desired, sequencesthat enhance protein secretion. The nature of such control sequencesdiffers depending upon the host organism; in prokaryotes, such controlsequences generally include a promoter, a ribosomal binding site, and atranscription termination sequence; in eukaryotes, generally, suchcontrol sequences include a promoter and a transcription terminationsequence. The term “control sequences” is intended to include componentswhose presence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

“Transformation.” refers to any process by which exogenous nucleic acid(e.g., a DNA molecule) enters a host cell. Transformation may occurunder natural or artificial conditions using various methods well knownin the art. Transformation may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method is selected based on the host cellbeing transformed and may include, but is not limited to, plasmid uptakeacross a cellular membrane, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), is intended torefer to a cell into which exogenous DNA has been introduced. In anembodiment, the host cell comprises two or more (e.g., multiple) nucleicacids encoding antibodies, such as, by way of non-limiting example, thehost cells described in U.S. Pat. No. 7,262,028. Such terms are intendedto refer not only to the particular subject cell, but, also to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. In an embodiment, host cells include prokaryotic andeukaryotic cells selected from any of the Kingdoms of life. In anotherembodiment, eukaryotic cells include protist, fungal, plant and animalcells. In another embodiment, host cells include but are not limited toprokaryotic species, such Escherichia coli; mammalian cell lines, suchas CHO, HEK 293, COS, NS0, SP2, and PER.C6; the insect cell line Sf9;and fungal cell species, such as Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, second ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1989).

“Transgenic organism.” as known in the art, refers to an organism havingcells that contain a transgene, wherein the transgene introduced intothe organism (or an ancestor of the organism) expresses a polypeptidenot naturally expressed in the organism. A “transgene” is a DNAconstruct, which is stably and operably integrated into the genome of acell from which a transgenic organism develops, directing the expressionof an encoded gene product in one or more cell types or tissues of thetransgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of hDLL4).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of hDLL4). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies have been described. See. e.g., PCT Publication No.WO01/83525.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist. Particular agonists of interest may include, but are notlimited to, members of the Notch-signaling pathway. DLL4 polypeptidesand nucleic acids, carbohydrates, or any other molecules that bind toDLL4.

The term “antagonist” or “inhibitor”, as used herein, refers to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological or immunologicalactivity of DLL4, especially human DLL4 (hDLL4). Antagonists andinhibitors of hDLL4 may include, but are not limited to, proteins,nucleic acids, carbohydrates, or any other molecule, which binds tohDLL4 and/or rodent DLL4.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof; inhibit orprevent the advancement of a disorder: cause regression of a disorder;inhibit or prevent the recurrence, development, onset, or progression ofone or more symptoms associated with a disorder; detect a disorder; orenhance or improve the prophylactic or therapeutic effect(s) of anothertherapy (e.g., prophylactic or therapeutic agent).

“Patient” and “subject” may be used interchangeably herein to refer toan animal, such as a mammal, including a primate (for example, a human,a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, acamel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guineapig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g., a duck ora goose), and a shark. Preferably, a patient or subject is a human, suchas a human being treated or assessed for a disease, disorder, orcondition; a human at risk for a disease, disorder, or condition: ahuman having a disease, disorder, or condition; and/or human beingtreated for a disease, disorder, or condition. More preferably, apatient or subject is being treated or assessed for cancer or otherdisease in which the existing aberrant DLL4 expression supports thecancer or other disease and inhibition or disruption of DLL4 activity isdesirable to treat the cancer or other disease.

The term “sample.” as used herein, is used in its broadest sense. A“biological sample,” as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, (e.g., whole blood), plasma, serum,urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

“Component,” “components.” and “at least one component.” refer generallyto a capture antibody, a detection or conjugate antibody, a control, acalibrator, a series of calibrators, a sensitivity panel, a container, abuffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, adetection reagent, a pretreatment reagent/solution, a substrate (e.g.,as a solution), a stop solution, and the like that can be included in akit for assay of a test sample, such as a patient urine, serum or plasmasample, in accordance with the methods described herein and othermethods known in the art. Thus, in the context of the presentdisclosure. “at least one component,” “component,” and “components” caninclude a polypeptide or other analyte as above, such as a compositioncomprising an analyte such as a polypeptide, which is optionallyimmobilized on a solid support, such as by binding to an anti-analyte(e.g., anti-polypeptide) antibody. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Risk” refers to the possibility or probability of a particular eventoccurring either presently or at some point in the future. “Riskstratification” refers to an array of known clinical risk factors thatallows physicians to classify patients into a low, moderate, high orhighest risk of developing a particular disease, disorder or condition.

“Specific” and “specificity” in the context of an interaction betweenmembers of a specific binding pair (e.g., an antigen or fragment thereofand an antibody or antigen binding fragment thereof) refer to theselective reactivity of the interaction. The phrase “specifically bindsto” and analogous phrases refer to the ability of antibodies (or antigenbinding fragments thereof) to bind specifically to a molecule ofinterest (or a fragment thereof) and not bind specifically to otherentities.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs,other specific binding pairs can include biotin and avidin (orstreptavidin), carbohydrates and lectins, complementary nucleotidesequences, effector and receptor molecules, cofactors and enzymes,enzyme inhibitors and enzymes, and the like. Furthermore, specificbinding pairs can include members that are analogs of the originalspecific binding members, for example, an analyte-analog. Immunoreactivespecific binding members include antigens, antigen fragments, andantibodies, including monoclonal and polyclonal antibodies as well ascomplexes, fragments, and variants (including fragments of variants)thereof, whether isolated or recombinantly produced.

“Variant” as used herein means a polypeptide that differs from a givenpolypeptide (e.g., DLL4 polypeptide or anti-DLL4 antibody) in amino acidsequence by the addition (e.g., insertion), deletion, or conservativesubstitution of amino acids, but that retains the biological activity ofthe given polypeptide (e.g., a variant DLL4 may compete with a wildtypeDLL4 for binding with an anti-DLL4 antibody if the variant DLL4 retainsthe original antibody binding site (epitope) of the wildtype DLL4). Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see. e.g., Kyte et al., J.Mol. Biol., 157: 105-132 (1982)). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of 2 are substituted. Thehydrophilicity of amino acids also can be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.4,554,101). Substitution of amino acids having similar hydrophilicityvalues can result in peptides retaining biological activity, for exampleimmunogenicity, as is understood in the art. In one aspect,substitutions are performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hydrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.“Variant” also can be used to describe a polypeptide or fragment thereofthat has been differentially processed, such as by proteolysis,phosphorylation, or other post-translational modification, yet retainsits biological activity or antigen reactivity. e.g., the ability to bindto DLL4. Use of “variant” herein is intended to encompass fragments of avariant unless otherwise contradicted by context.

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, serum, urine, synovial fluid, cells,organs, tissues, bone marrow, lymph nodes, and spleen.

I. Antibodies that Bind Human DLL4.

One aspect of the present invention provides isolated human monoclonalantibodies, or antigen-binding portions thereof, that bind to DLL4 withhigh affinity, a slow off rate, and/or high neutralizing capacity.Advantageously, such human antibodies or antigen-binding portionsthereof that bind DLL4 find use as human therapeutic agents that can beadministered to a human patient with minimal or no response by thepatient's immune system toward the administered therapeutic DLL4 bindingprotein. Accordingly, a patient may obtain the benefit of such fullyhuman DLL4 binding proteins over the course of repeated administrations.Other aspects of the invention provide chimeric antibodies that bindDLL4 and CDR grafted antibodies, or antigen-binding portions thereof,that bind DLL4. Preferably, the antibodies, or portions thereof, areisolated antibodies. Preferably, the antibodies of the invention areneutralizing human anti-DLL4 antibodies.

A. Method of Making Anti DLL4 Antibodies.

Antibodies of the present invention may be made by any of a number oftechniques known in the art. A preferred method is PROfusion mRNAdisplay technology as exemplified in Example 2 herein. Another method isto immunize a transgenic rodent (e.g., a transgenic mouse) that carriesa functional complement of human immunoglobulin genes with human DLL4 orantigenic portion thereof followed by standard hybridoma technology togenerate hybridomas that express fully human monoclonal antibodies thatbind human DLL4. Recombinant human antibodies obtain by such a methodhave variable and constant regions derived from human germlineimmunoglobulin sequences. Such methods provide fully human DLL4 bindingproteins and eliminate the need to otherwise carry out one or morerounds of humanization to reduce the sources of non-human antigenicityof the monoclonal DLL4 antibody molecules. Accordingly, techniquesutilizing material from multiple species are less preferred but may beused.

It is also noted that the term “monoclonal antibody” as used herein isnot limited to antibodies produced through hybridoma technology. Theterm “monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced.

Additional aspects of various techniques that may be employed to obtainDLL4 monoclonal antibody molecules according to the invention aredescribed below.

1. Anti-DLL4 Monoclonal Antibodies Using Hybridoma Technology.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, second edition, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988); Hammerling, et al., In MonoclonalAntibodies and T-Cell Hybridomas, pages 563-681 (Elsevier, New York,1981) (said references incorporated by reference in their entireties).Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention. Briefly, mice can be immunized withan DLL4 antigen. In a preferred embodiment, the DLL4 antigen isadministered with a adjuvant to stimulate the immune response. Suchadjuvants include complete or incomplete Freund's adjuvant, RIBI(muramyl dipeptides) or ISCOM (immunostimulating complexes). Suchadjuvants may protect the polypeptide from rapid dispersal bysequestering it in a local deposit, or they may contain substances thatstimulate the host to secrete factors that are 15 chemotactic formacrophages and other components of the immune system. Preferably, if apolypeptide is being administered, the immunization schedule willinvolve two or more administrations of the polypeptide, spread out overseveral weeks; however, a single administration of the polypeptide mayalso be used.

After immunization of an animal with a DLL4 antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-DLL4antibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-DLL4 antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen DLL4 are detected in the mouse serum, the mouse spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the American Type Culture Collection(ATCC. Manassas, Va., US). Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding DLL4. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using DLL4, or a portion thereof,or a cell expressing DLL4. In a preferred embodiment, the initialscreening is performed using an enzyme-linked immunosorbent assay(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example ofELISA screening is provided in PCT Publication No. WO 00/37504,incorporated herein by reference.

Anti-DLL4 antibody-producing hybridomas are selected, cloned, andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production, and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system. e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

In a preferred embodiment, hybridomas are mouse hybridomas, as describedabove. In another embodiment, hybridomas are produced in a non-human,non-mouse species such as rats, sheep, pigs, goats, cattle, or horses.In yet another preferred embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-DLL4 antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab)2 fragments of the inventionmay be produced by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab)2 fragments). F(ab′)2 fragments contain the variableregion, the light chain constant region, and the CH1 domain of the heavychain.

2. Anti-DLL4 Monoclonal Antibodies Using SLAM.

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook etal., Proc. Natl. Acad Sci. USA, 93: 7843-7848 (1996). In this method,single cells secreting antibodies of interest. e.g., lymphocytes derivedfrom any one of the immunized animals described in Section I.A.1(above), are screened using an antigen-specific hemolytic plaque assay,wherein the antigen DLL4, a subunit of DLL4, or a fragment thereof, iscoupled to sheep red blood cells using a linker, such as biotin, andused to identify single cells that secrete antibodies with specificityfor DLL4. Following identification of antibody-secreting cells ofinterest, heavy- and light-chain variable region cDNAs are rescued fromthe cells by reverse transcriptase-PCR (RT-PCR) and these variableregions can then be expressed, in the context of appropriateimmunoglobulin constant regions (e.g., human constant regions), inmammalian host cells, such as COS or CHO cells. The host cellstransfected with the amplified immunoglobulin sequences, derived from invivo selected lymphocytes, can then undergo further analysis andselection in vitro, for example by panning the transfected cells toisolate cells expressing antibodies to DLL4. The amplifiedimmunoglobulin sequences further can be manipulated in vitro, such as byin vitro affinity maturation method. See, for example, PCT PublicationNo. WO 97/29131 and PCT Publication No. WO 00/56772.

3. Anti-DLL4 Monoclonal Antibodies Using Transgenic Animals.

In another embodiment of the instant invention, antibodies are producedby immunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with an DLL4 antigen. In a preferred embodiment,the non-human animal is a XENOMOUSE® transgenic mouse, an engineeredmouse strain that comprises large fragments of the human immunoglobulinloci and is deficient in mouse antibody production. See, e.g., Green etal., Nature Genetics, 7: 13-21 (1994) and U.S. Pat. Nos. 5,916,771;5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598; and6,130,364. See also PCT Publication Nos. WO 91/10741; WO 94/02602; WO96/34096; WO 96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO99/45031; WO 99/53049; WO 00/09560; and WO 00/37504. The XENOMOUSE®transgenic mouse produces an adult-like human repertoire of fully humanantibodies, and generates antigen-specific human monoclonal antibodies.The XENOMOUSE® transgenic mouse contains approximately 80% of the humanantibody repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics, 15: 146-156 (1997),Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the disclosuresof which are hereby incorporated by reference.

4. Anti-DLL4 Monoclonal Antibodies Using Recombinant Antibody Libraries.

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired DLL4-binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, U.S. Pat. No. 5,223,409(Ladner et al.); PCT Publication No. WO 92/18619 (Kang et al.); PCTPublication No. WO 91/17271 (Dower et al.); PCT Publication No. WO92/20791 (Winter et al.); PCT Publication No. WO 92/15679 (Markland etal.); PCT Publication No. WO 93/01288 (Breitling et al.); PCTPublication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO92/09690 (Garrard et al.): Fuchs et al., Bio/Technology, 9: 1369-1372(1991): Hay et al., Hum. Antibod. Hybridomas, 3: 81-85 (1992); Huse etal., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkinset al., J. Mol. Biol., 226: 889-896 (1992); Clackson et al., Nature,352: 624-628 (1991); Gram et al., Proc. Natl. Acad Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio/Technology, 9: 1373-1377 (1991);Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991); Barbas etal., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); US PatentApplication Publication No. 2003/0186374; and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

The recombinant antibody library may be from a subject immunized withDLL4, or a portion of DLL4. Alternatively, the recombinant antibodylibrary may be from a naïve subject, i.e., one who has not beenimmunized with DLL4, such as a human antibody library from a humansubject who has not been immunized with human DLL4. Antibodies of theinvention are selected by screening the recombinant antibody librarywith the peptide comprising human DLL4 to thereby select thoseantibodies that recognize DLL4. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for DLL4, such as thosethat dissociate from human DLL4 with a particular K_(off) rate constant,the art-known method of surface plasmon resonance can be used to selectantibodies having the desired K_(off) rate constant. To selectantibodies of the invention having a particular neutralizing activityfor hDLL4, such as those with a particular IC₅₀, standard methods knownin the art for assessing the inhibition of DLL4 activity may be used.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human DLL4. Preferably, theantibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody. Forexample, the antibodies of the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.Such phage can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phageincluding fd and M13 binding domains expressed from phage with Fab, Fv,or disulfide stabilized Fv antibody domains recombinantly fused toeither the phage gene III or gene VIII protein. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include those disclosed in Brinkmann et al., J. Immunol.Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods,184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24: 952-958(1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al., Advancesin Immunology, 57: 191-280 (1994); PCT Publication No. WO 92/01047; PCTPublication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and5,969,108: each of which is incorporated herein by reference in itsentirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′, and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324: Mullinax et al., BioTechniques, 12(6):864-869 (1992): Sawai et al., Am. J. Reprod. Immunol., 34: 26-34 (1995);and Better et al., Science, 240: 1041-1043 (1988) (said referencesincorporated by reference in their entireties). Examples of techniqueswhich can be used to produce single-chain Fvs and antibodies includethose described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston etal., Methods in Enzymology, 203: 46-88 (1991); Shu et al., Proc. Natl.Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al., Science, 240:1038-1041 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification ofantibodies of the invention. One type of alternative expression systemis one in which the recombinant antibody library is expressed asRNA-protein fusions, as described in PCT Publication No. WO 98&31700(Szostak and Roberts), and in Roberts and Szostak, Proc. Natl. Acad.Sci. USA, 94:12297-12302 (1997). In this system, a covalent fusion iscreated between an mRNA and the peptide or protein that it encodes by invitro translation of synthetic mRNAs that carry puromycin, a peptidylacceptor antibiotic, at their 3′ end. Thus, a specific mRNA can beenriched from a complex mixture of mRNAs (e.g., a combinatorial library)based on the properties of the encoded peptide or protein, e.g.,antibody, or portion thereof, such as binding of the antibody, orportion thereof, to the dual specificity antigen. Nucleic acid sequencesencoding antibodies, or portions thereof, recovered from screening ofsuch libraries can be expressed by recombinant means as described above(e.g., in mammalian host cells) and, moreover, can be subjected tofurther affinity maturation by either additional rounds of screening ofmRNA-peptide fusions in which mutations have been introduced into theoriginally selected sequence(s), or by other methods for affinitymaturation in vitro of recombinant antibodies, as described above. Apreferred example of this methodology, is the PROfusion displaytechnology employed in the Examples (infra).

In another approach the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e, g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed in U.S. Pat.No. 6,699,658 (Wittrup et al.) incorporated herein by reference.

B. Production of Recombinant DLL4 Antibodies

Antibodies of the present invention may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the heavy and light chains is(are) transfected into a host cell by standard techniques. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NS0myeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr− CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

1. Anti-DLL4 Antibodies.

Amino acid sequences of VH and VL regions of isolated fully humanantibodies that bind human DLL4 are shown for clones E9 and A10 in Table4 (See. Examples, below). The isolated anti-DLL4 antibody CDR sequencesof the E9 and A10 antibodies establish two novel families of DLL4binding proteins, isolated in accordance with this invention, andcomprising polypeptides that include the CDR sequences derived from E9and affinity matured clones thereof or derived from A10 and affinitymatured clones thereof. The variable regions and CDRs of the E9monoclonal antibody and affinity matured derivatives thereof are listedin Tables 4, 8, 14, 18, and 19. The variable regions and CDRs of the A10monoclonal antibody and affinity matured derivatives thereof are listedin Table 4, 9, and 10. To generate and to select CDRs for bindingproteins according to the invention having preferred DLL4 binding and/orneutralizing activity with respect to human DLL4, standard methods knownin the art for generating binding proteins of the present invention andassessing the DLL4 binding and/or neutralizing characteristics of thosebinding protein may be used, including but not limited to thosespecifically described herein.

Based on an alignment of the amino acid sequences of the CDRs of theheavy chain variable regions (VH) and the light chain variable regions(VL) of the anti-DLL4 antibody E9 clones described herein, the inventionprovides a DLL4 binding protein comprising an antigen binding domaincapable of binding human DLL4, said antigen binding domain comprising atleast one or more CDRs selected from the group consisting of:

(SEQ ID NO: 99) CDR-H1: X₁-X₂-X₃-X₄-X₅-X₆-X_(7,)wherein;

-   -   X₁ is S or N;    -   X₂ is S. G or N;    -   X₃ is S, N, T, G or R;    -   X₄ is Y;    -   X₅ is Y or H;    -   X₆ is W; and    -   X₇ is G;

(SEQ ID NO: 100) CDR-H2: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X_(16,)wherein;

-   -   X₁ is D;    -   X₂ is I;    -   X₃ is Y, N, or S;    -   X₄ is Y;    -   X₅ is T, N, A, I, S or R;    -   X₆ is G;    -   X₇ is S, N, T or G;    -   X₈ is T;    -   X₉ is Y;    -   X₁₀ is Y;    -   X₁₁ is N;    -   X₁₂ is P;    -   X₁₃ is S;    -   X₁₄ is L;    -   X₁₅ is K; and    -   X₁₆ is S, N, D or G;

(SEQ ID NO: 101) CDR-H3: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X_(11,)wherein;

-   -   X₁ is E, Y, F, Q, W, L, or A;    -   X₂ is D, A, S, G, V, E or N;    -   X₃ is V, M, L, P, or A;    -   X₄ is I, A, P, R, S, K, Q, V, G, M or E;    -   X₅ is L, Y, F or M;    -   X₆ is R, G, S, Q or A    -   X₇ is G;    -   X₈ is G, A or S;    -   X₉ is S, A, L, V, R or G;    -   X₁₀ is D; and    -   X₁₁ is Y, D, S, N, H, E, R, L, P, C, 1 M, T, Q, or K;

(SEQ ID NO: 102) CDR-L1: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X_(11,)wherein;

-   -   X₁ is S;    -   X₂ is G;    -   X₃ is Q, E or D;    -   X₄ is R, S, G, M, K, L or T;    -   X₅ is L;    -   X₆ is G;    -   X₇ is D or E;    -   X₈ is K;    -   X₉ is Y;    -   X₁₀ is A or V; and    -   X₁₁ is S;

(SEQ ID NO: 103) CDR-L2: X₁-X₂-X₃-X₄-X₅-X₆-X_(7,)wherein;

-   -   X₁ is E or Q;    -   X₂ is D;    -   X₃ is S, L, T, A, E or F;    -   X₄ is K, T, E, N, Q, S, or M;    -   X₅ is R;    -   X₆ is P; and    -   X₇ is S;        and

(SEQ ID NO: 104) CDR-L3: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X_(9,)wherein:

-   -   X₁ is Q;    -   X₂ is A;    -   X₃ is W;    -   X₄ is D;    -   X₅ is R, S, M, E, N, G, or K;    -   X₆ is D or E    -   X₇ is T, V, A, S or M;    -   X₈ is G, A or C; and    -   X₉ is V.

Preferably, a DLL4 binding protein comprising one or more CDRs describedabove binds human (“hu”, “h”) DLL4 and also one or more DLL4 proteinsselected from the group consisting of: mouse (“murine”, “mu”) DLL4,cynomolgus monkey (“cynomolgus”. “cyno”) DLL4, and rat DLL4.

Based on an alignment of the amino acid sequences of the CDRs of theheavy chain variable regions (VH) and the light chain variable regions(VL) of the anti-DLL4 antibody A10 clones described herein, theinvention provides a DLL4 binding protein comprising an antigen bindingdomain capable of binding human DLL4, said antigen binding domaincomprising at least one or more CDRs selected from the group consistingof:

(SEQ ID NO: 105) CDR-H1: X₁-X₂-X₃-X₄-X_(5,)wherein;

-   -   X₁ is S, N, or D;    -   X₂ is H or Y;    -   X₃ is W;    -   X₄ is M; and    -   X₅ is S or H;

(SEQ ID NO: 106) CDR-H2: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X_(17,)wherein;

-   -   X₁ is I, D, M, or T;    -   X₂ is I;    -   X₃ is S;    -   X₄ is Y, N, S, Q, V, T, H, or D;    -   X₃ is D;    -   X₆ is G;    -   X₇ is S, R, I, T, G, K, H, or N;    -   X₈ is N, Y, S, I, or T;    -   X₉ is K, M, N, Q, E, T, R, S, A, or L;    -   X₁₀ is Y, D, or E;    -   X₁₁ is S or Y;    -   X₁₂ is A;    -   X₁₃ is D;    -   X₁₄ is S;    -   X₁₅ is V;    -   X₁₆ is K; and    -   X₁₇ is G;

(SEQ ID NO: 107) CDR-H3: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X_(10,)wherein:

-   -   X₁ is A;    -   X₂ is G, A, or R;    -   X₃ is G;    -   X₄ is G, S, or A;    -   X₅ is N;    -   X₆ is V or M;    -   X₇ is G;    -   X₈ is F, L, Y, or M;    -   X₉ is D; and    -   X₁₀ is I, S, or L;

CDR-L1: (SEQ ID NO: 108) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁,wherein:

-   -   X₁ is S;    -   X₂ is A or G;    -   X₃ is D;    -   X₄ is K, N, L, Q, M, E, S, T, G, or D;    -   X₅ is L.    -   X₆ is G;    -   X₇ is T, S, N, A, G, or E;    -   X₈ is K, Q, N, or R;    -   X₉ is Y;    -   X₁₀ is V or I; and    -   X₁₁ is S;

CDR-L2: (SEQ ID NO: 109) X₁-X₂-X₃-X₄-X₅-X₆-X₇,wherein;

-   -   X₁ is Q;    -   X₂ is D;    -   X₃ is A, G. W. S, or D;    -   X₄ is K, M, Q, N, L, T, I, or E;    -   X₅ is R;    -   X₆ is P; and    -   X₇ is S;        and

CDR-L3: (SEQ ID NO: 110) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉,wherein;

-   -   X₁ is Q;    -   X₂ is S or A;    -   X₃ is W;    -   X₄ is D;    -   X₅ is R, S, Q, P, A, V, W, or M;    -   X₆ is S, G, I, N, R, or T    -   X₇ is D or G;    -   X₈ is V, A, P, or E; and    -   X₉ is V.

Preferably, a DLL4 binding protein comprising one or more CDRs describedabove binds human (“hu”) DLL4 and also cynomolgus monkey (“cynomolgus”,“cyno”) DLL4.

2. Anti-DLL4 Chimeric Antibodies.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. See e.g., Morrison, Science, 229:1202-1207 (1985). Oi et al., BioTechniques, 4: 214 (1986): Gillies etal., J. Immunol. Methods, 125: 191-202 (1989): U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entireties. In addition, techniques developed for the productionof “chimeric antibodies” by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.See, for example, Morrison et al., Proc. Natl. Acad Sci. USA, 81:6851-6855 (1984); Neuberger et al., Nature, 312: 604-608 (1984); Takedaet al., Nature, 314: 452-454 (1985), which are incorporated herein byreference in their entireties.

3. Anti-DLL4 CDR Grafted Antibodies.

The isolated anti-DLL4 antibody CDR sequences of the invention may beused to make CDR-grafted antibodies to modulate the properties of theoriginal antibody. Such properties include but are not limited tobinding kinetics, affinity, biological activities, speciescross-reactivity, molecule cross-reactivity, epitope, physicochemicalproperties, pharmacokinetic properties, pharmacodynamic properties, orpharmacological properties. CDR-grafted antibodies comprise heavy andlight chain variable region sequences from a human antibody or anon-human primate antibody wherein one or more of the CDR regions of VHand/or VL are replaced with CDR sequences of the original anti-DLL4antibody. A framework sequence from any human or non-human primateantibody may serve as the template for CDR grafting. However, straightchain replacement onto such a framework often leads to some loss ofbinding affinity to the antigen. The more homologous a human, or otherspecies, antibody is to the original human antibody, the less likely thepossibility that combining the CDRs with the new human framework ornon-human primate framework will introduce distortions in the CDRs thatcould reduce affinity or other properties. Therefore, it is preferablethat the variable framework that is chosen to replace the human variableregion framework apart from the CDRs has at least a 30% sequenceidentity with the human antibody variable region framework. It is morepreferable that the variable region framework that is chosen to replacethe human variable region framework apart from the CDRs has at least a40% sequence identity with the human antibody variable region framework.It is more preferable that the variable region framework that is chosento replace the human variable framework apart from the CDRs has at leasta 50% sequence identity with the human antibody variable regionframework. It is more preferable that the variable region framework thatis chosen to replace the human variable framework apart from the CDRshas at least a 60% sequence identity with the human antibody variableregion framework. It is more preferable that the new human or non-humanprimate and the original human variable region framework apart from theCDRs has at least 70% sequence identity. It is even more preferable thatthe new human or non-human primate and the original human variableregion framework apart from the CDRs has at least 75% sequence identity.It is most preferable that the new human or non-human primate and theoriginal human variable region framework apart from the CDRs has atleast 80% sequence identity. Even using a highly homologous human ornon-human primate framework to graft CDRs of the original humananti-DLL4 antibody, the resulting grafted antibody may still losebinding affinity to antigen to some degree. In this case, to regain theaffinity it is necessary to include at least one or more key frameworkresidue(s) substitution of the original antibody to the correspondingposition of the newly grafted antibody. Such a key residue may beselected from the group consisting of:

-   -   a residue adjacent to a CDR;    -   a glycosylation site residue;    -   a rare residue;    -   a residue capable of interacting with human DLL4    -   a canonical residue;    -   a contact residue between heavy chain variable region and light        chain variable region;    -   a residue within a Vernier zone; and    -   a residue in a region that overlaps between a Chothia-defined        variable heavy chain CDR1 and a Kabat-defined first heavy chain        framework.

4. Anti-DLL4 Humanized Antibodies.

While the compositions of the present invention eliminate therequirement to make humanized antibodies, humanized DLL4 antibodies maybe prepared using compositions of the invention. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementary determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Known human Ig sequences are disclosed at websites available via the world wide web (www.), e.g.,ncbi.nlm.nih.gov/entrez/query.fcgi; atcc.org/phage/hdb.html;sciquest.com/; abcam.com/; antibodyresource.com/onlinecomp.html;public.iastate.edu/.about.pedro-/research_tools.html;mgen.uniheidelberg.de/SD/IT/IT.html;whfreeman.com/immunology-/CH05/kuby05.htm;library.thinkquest.org/12429/Immune/Antibody.html;hhmi.org/grants/lectures/1996/vlab/;path.-cam.ac.uk/.about.mrc7/mikeimages.html; antibodyresource.com/;mcb.harvard.edu/BioLinks-/Immunology.html; immunologylink.com/;pathbox.wustl.edu/.about.hcenter/index.html;bio-tech.ufl.edu/.about.hcl/; pebio.com/pa/340913-/340913.html;nal.usda.gov/awic/pubs/antibody/;m.ehimeu.acjp/.about.yasuhito-/Elisa.html; biodesign.com/table.asp;icnet.uk/axp/facs/davies/links.html;biotech.ufl.edu-/.about.fccl/protocol.html; isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rekiAEP-Start.html;baserv.uci.kun.nl/.about.jraats/linksl.html;recab.uni-hd.de/immuno.bme.nwu.edu/;mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;ibt.unam.mx/-vir/V_mice.html; imgt.cnusc.fr:8104/;biochem.ucl.ac.uk/.about.martin/abs/index.html; anti-body.bath.ac.uk/;abgen.cvm.tamu.edulab/wwwabgen.html;unizh.ch/.about.honegger/AHO-seminar/Slide01.html;cryst.bbk.ac.uk.about.ubcg07s/; nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;ibt.unam.mx/vir/structure/stat_aim.-html;biosci.missouri.edu/smithgp/index.html;cryst.bioc.cam.ac.uk/.about.fmolina/Webpages-/Pept/spottech.html;jerini.de/frroducts.htm; patents.ibm.com/ibm.html. Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art. e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089 (Queen et al.); Riechmann et al., Nature, 332:323-327 (1988), which are incorporated herein by reference in theirentireties.) Three-dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Antibodies can be humanized using a varietyof techniques known in the art, such as but not limited to thosedescribed in Jones et al., Nature, 321: 522-525 (1986); Verhoeyen etal., Science, 239: 1534-1536 (1988), Sims et al., J. Immunol., 151:2296-2308 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987),Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992); Prestaet al., J. Immunol., 151: 2623-2632 (1993), Padlan, E. A., MolecularImmunology, 28(4/5): 489-498 (1991); Studnicka et al., ProteinEngineering, 7(6): 805-814 (1994): Roguska, et al., Proc. Natl. AcadSci. USA, 91:969-973 (1994): PCT Publication Nos. WO 91/09967. WO99/06834 (PCT/US98/16280). WO 97/20032 (PCT/US96/18978), WO 92/11272(PCT/US91/09630). WO 92/03461 (PCT/US91/05939). WO 94/18219(PCT/US94/01234). WO 92/01047 (PCT/GB91/01134), WO 93/06213(PCT/GB92/01755), WO90/14443, WO90/14424, and WO90/14430; EuropeanPublication Nos. EP 0 592 106, EP 0 519 596, and EP 0 239 400; U.S. Pat.Nos. 5,565,332; 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567, each entirelyincorporated herein by reference, included references cited therein.

C. Production of Antibodies and Antibody-Producing Cell Lines.

Preferably, anti-DLL4 antibodies of the present invention exhibit a highcapacity to reduce or to neutralize tumor angiogenesis activity, e.g.,as assessed by any one of several in vitro and in vivo assays known inthe art. Evaluating the neutralization of activity of DLL4 can beassessed via several in vitro and in vivo assays know in the art.Exemplary parameters for assessing neutralization of DLL4 activityinclude, but are not limited to, antibodies that inhibit DLL4interaction with the Notch receptor, and/or Notch-signaling pathway withan IC₅₀ values of about at least 10⁻⁶ M; at least 10⁻⁷ M, or at least10⁻⁸ M.

Preferably, anti-DLL4 antibodies of the present invention also exhibit ahigh capacity to reduce or to neutralize DLL4 activity.

In preferred embodiments, the isolated antibody, or antigen-bindingportion thereof, binds human DLL4, wherein the antibody, orantigen-binding portion thereof, dissociates from human DLL4 with aK_(off) rate constant of about 0.1 s⁻¹ or less, as determined by surfaceplasmon resonance, or which inhibits DLL4 and/or human DLL4 activitywith an IC₅₀ of about 1×10⁻⁶M or less. Alternatively, the antibody, oran antigen-binding portion thereof, may dissociate from human DLL4 witha K_(off) rate constant of about 1×10⁻²s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human DLL4 and/or human DLL4activity with an IC₅₀ of about 1×10⁻⁷ M or less. Alternatively, theantibody, or an antigen-binding portion thereof, may dissociate fromhuman DLL4 with a K_(off) rate constant of about 1×10⁻³s⁻¹ or less, asdetermined by surface plasmon resonance, or may inhibit human DLL4 withan IC₅₀ of about 1×10⁻⁸M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human DLL4 with aK_(off) rate constant of about 1×10⁻⁴s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit DLL4 activity with an IC₅₀ ofabout 1×10⁻⁹M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human DLL4 with aK_(off) rate constant of about 1×10⁻⁵s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit DLL4 and/or human DLL4activity with an IC₅₀ of about 1×10⁻¹⁰M or less. Alternatively, theantibody, or an antigen-binding portion thereof, may dissociate fromhuman DLL4 with a K_(off) rate constant of about 1×10⁻⁵s⁻¹ or less, asdetermined by surface plasmon resonance, or may inhibit DLL4 and/orhuman DLL4 activity with an IC₅₀ of about 1×10⁻¹¹M or less.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgDconstant region. Preferably, the heavy chain constant region is an IgG1heavy chain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (see, U.S. Pat. Nos. 5,648,260and 5,624,821 (Winter et al.)). The Fc portion of an antibody mediatesseveral important effector functions e.g. cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC), andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered.

One embodiment provides a labeled binding protein wherein an antibody orantibody portion of the invention is derivatized or linked to anotherfunctional molecule (e.g., another peptide or protein). For example, alabeled binding protein of the invention can be derived by functionallylinking an antibody or antibody portion of the invention (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

Another embodiment of the invention provides a crystallized DLL4 bindingprotein. Preferably, the invention relates to crystals of DLL4 bindingproteins described herein, including whole anti-DLL4 antibodies,fragments thereof, as well as antibody constructs and binding proteinconjugates (including antibody conjugates) as disclosed herein, andformulations and compositions comprising such crystals. In oneembodiment, the crystallized binding protein has a greater half-life invive than the soluble counterpart of the binding protein. In anotherembodiment the binding protein retains biological activity aftercrystallization. Crystallized binding proteins of the invention may beproduced according methods known in the art and as disclosed in PCTPublication No. WO 02/72636, incorporated herein by reference.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Protein glycosylation depends onthe amino acid sequence of the protein of interest, as well as the hostcell in which the protein is expressed. Different organisms may producedifferent glycosylation enzymes (e.g., glycosyltransferases andglycosidases), and have different substrates (nucleotide sugars)available. Due to such factors, protein glycosylation pattern, andcomposition of glycosyl residues, may differ depending on the hostsystem in which the particular protein is expressed. Glycosyl residuesuseful in the invention may include, but are not limited to, glucose,galactose, mannose, fucose, n-acetylglucosamine and sialic acid.Preferably the glycosylated binding protein comprises glycosyl residuessuch that the glycosylation pattern is human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (US Patent ApplicationPublication Nos. 2004/0018590 and 2002/0137134).

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

D. Uses of DLL4 Binding Proteins.

Given their ability to bind to human DLL4 and murine DLL4, the DLL4binding proteins described herein, including antibodies and portionsthereof, can be used to detect or measure DLL4 in a sample (e.g., in amixture, solution, or biological sample, such as blood, serum, orplasma), using any of the conventional immunoassays known in the art,such as an enzyme linked immunosorbent assays (ELISA), aradioimmunoassay (RIA), or a tissue immunohistochemistry. The inventionprovides a method for detecting human DLL4 and/or murine DLL4 in asample comprising contacting a sample with a DLL4 binding protein anddetecting either the DLL4 binding protein bound to human DLL4 and/ormurine DLL4 or the unbound binding protein to thereby detect human DLL4and/or murine DLL4 in the sample. A DLL4 binding protein describedherein can be directly or indirectly labeled with a detectable substanceto facilitate detection of the bound or unbound DLL4 binding protein.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, (5-galactosidase, or acetylcholinesterase:examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ³H, ¹⁴C,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Biological samples that can be assayed for DLL4 include urine, feces,blood, serum, plasma, perspiration, saliva, oral swab (cheek, tongue,throat), vaginal swab, rectal swab, dermal swab, dermal scrape, tissuebiopsy, as well as any other tissue sample that can be obtained bymethods available in the art.

Alternative to labeling the binding protein, human DLL4 can be assayedin biological fluids by a competition immunoassay utilizing recombinanthuman (rh) DLL4 standards labeled with a detectable substance and anunlabeled DLL4 binding protein described herein. In this assay, thebiological sample, the labeled rhDLL4 standards, and the DLL4 bindingprotein are combined and the amount of labeled rhDLL4 standard bound tothe unlabeled binding protein is determined. The amount of human DLL4 inthe biological sample is inversely proportional to the amount of labeledrhDLL4 standard bound to the DLL4 binding protein. Similarly, human DLL4can also be assayed in biological fluids by a competition immunoassayutilizing rhDLL4 standards labeled with a detectable substance and anunlabeled DLL4 binding protein described herein.

The DLL4 binding proteins of the invention preferably are capable ofneutralizing DLL4 activity, in particular hDLL4 activity, both in vitroand in vivo. Accordingly, such binding proteins of the invention can beused to inhibit DLL4 activity. e.g., in a cell culture containing DLL4,in human subjects, or in other mammalian subjects expressing a DLL4 withwhich a binding protein of the invention cross-reacts. In oneembodiment, the invention provides a method for inhibiting DLL4 activitycomprising contacting a DLL4 with a DLL4 antibody or antibody portion ofthe invention such that DLL4 activity is inhibited. For example, in acell culture containing or suspected of containing DLL4, an antibody orantibody portion of the invention can be added to the culture medium toinhibit DLL4 activity in the culture.

In another embodiment, the invention provides a method for reducing DLL4activity in a subject, advantageously from a subject suffering from adisease or disorder in which DLL4 or DLL4 activity is detrimental. Theinvention provides methods for reducing DLL4 or DLL4 activity in asubject suffering from such a disease or disorder, which methodcomprises administering to the subject a DLL4 binding protein of theinvention such that DLL4 or DLL4 activity in the subject is reduced.Preferably, the DLL4 is human DLL4, and the subject is a human subject.Alternatively, the subject can be a mammal expressing a DLL4 to which aDLL4 binding protein of the invention is capable of binding. Stillfurther, the subject can be a mammal into which DLL4 has been introduced(e.g., by administration of DLL4 or by expression of a DLL4 transgene).An antibody or other DLL4 binding protein of the invention can beadministered to a human subject for therapeutic purposes. Moreover, aDLL4 binding protein of the invention can be administered to a non-humanmammal expressing a DLL4 with which the binding protein is capable ofbinding for veterinary purposes or as an animal model of human disease.Regarding the latter, such animal models may be useful for evaluatingthe therapeutic efficacy of antibodies and other DLL4 binding proteinsof the invention (e.g., testing of dosages and time courses ofadministration).

As used herein, the term “a disorder in which DLL4 and/or Notchsignaling activity is detrimental” is intended to include diseases, suchas cancer, and other disorders in which the presence of DLL4 and/orNotch signaling activity in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which DLL4 and/orNotch signaling activity is detrimental is a disorder in whichalteration of DLL4 and/or Notch signaling activity is expected toalleviate the symptoms and/or progression of the disorder (e.g., tumorgrowth). Such disorders may be evidenced, for example, by an increase inangiogenesis in a subject suffering from the disorder (e.g., an increasein the concentration of various proteins known in the art to increase inserum, plasma, synovial fluid, etc., of the subject during tumor growthand formation), which can be detected, for example, using an anti-DLL4antibody as described above. Non-limiting examples of disorders that canbe treated with the antibodies of the invention include those disordersdiscussed in the section below pertaining to pharmaceutical compositionsof the antibodies of the invention.

II. Pharmaceutical Compositions.

The invention also provides pharmaceutical compositions comprising aDLL4 binding protein of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical compositions comprising DLL4 bindingproteins of the invention are for use in, but not limited to,diagnosing, detecting, or monitoring a disorder; in preventing,treating, managing, or ameliorating a disorder or one or more symptomsthereof; and/or in research. In a specific embodiment, a compositioncomprises one or more DLL4 binding proteins of the invention. In anotherembodiment, the pharmaceutical composition comprises one or more bindingproteins of the invention and one or more prophylactic or therapeuticagents other than binding proteins of the invention for treating adisorder in which DLL4 and/or DLL4 activity is detrimental. Preferably,the prophylactic or therapeutic agents known to be useful for or havingbeen or currently being used in the prevention, treatment, management,or amelioration of a disorder, such as cancer or a tumor, or one or moresymptoms thereof. In accordance with these embodiments, the compositionmay further comprise of a carrier, diluent, or excipient.

The binding proteins of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises a DLL4 bindingprotein (or DLL4 binding portion thereof) of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore DLL4 binding proteins of the invention or the combination of one ormore binding proteins of the invention and a prophylactic agent ortherapeutic agent useful for preventing, managing, treating, orameliorating a disorder or one or more symptoms thereof, e.g., reducingtumor angiogenesis, encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the DLL4 bindingprotein, receptor-mediated endocytosis (see, e, g., Wu and Wu, J. Biol.Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of administering aprophylactic or therapeutic agent of the invention include, but are notlimited to, parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural administration,intratumoral administration, and mucosal administration (e.g.,intranasal and oral routes). In addition, pulmonary administration canbe employed, e.g., by use of an inhaler or nebulizer, and formulationwith an aerosolizing agent. See. e.g., U.S. Pat. Nos. 6,019,968;5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO97/44013, WO 98/31346, and WO 99/66903, each of which is incorporatedherein by reference their entireties. In one embodiment, a DLL4 bindingprotein of the invention, combination therapy, or a composition of theinvention is administered using Alkermes AIR® pulmonary drug deliverytechnology (Alkermes, Inc., Cambridge, Mass. US). In a specificembodiment, prophylactic or therapeutic agents of the invention areadministered intramuscularly, intravenously, intratumorally, orally,intranasally, pulmonary, or subcutaneously. The prophylactic ortherapeutic agents may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more DLL4 binding proteins ofthe invention antagonists is administered locally to the affected areato a subject to prevent, treat, manage, and/or ameliorate a disorder ora symptom thereof. In another embodiment, an effective amount of one ormore DLL4 binding proteins of the invention is administered locally tothe affected area in combination with an effective amount of one or moretherapies (e.g., one or more prophylactic or therapeutic agents) otherthan a binding protein of the invention of a subject to prevent, treat,manage, and/or ameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see, Langer (Science, 249: 1527-1533 (1990)); Sefton, CRC Crit.Ref. Biomed. Eng., 14: 201-240 (1987): Buchwald et al., Surgery, 88:507-516 (1980); Saudek et al., N. Engl. J. Med., 321: 574-579 (1989)).In another embodiment, polymeric materials can be used to achievecontrolled or sustained release of the therapies of the invention. See.e.g., Goodson, J. M, In Medical Applications of Controlled Release, Vol.II, Applications and Evaluations, (Langer and Wise, eds.), (CRC PressInc., Boca Raton, 1984), chapter 6, pages 115-138; Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.) (Wiley, New York, 1984): Langer and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. Phys., C23: 61-126 (1983); see also, Levy et al.,Science, 228: 190-192 (1985); During et al., Ann. Neurol., 25: 351-356(1989); Howard et al., J. Neurosurg., 71: 105-112 (1989); U.S. Pat. Nos.5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; and PCTPublication Nos. WO 99/15154 and WO 99/20253. Examples of polymers usedin sustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In apreferred embodiment, the polymer used in a sustained releaseformulation is inert, free of leachable impurities, stable on storage,sterile, and biodegradable. In yet another embodiment, a controlled orsustained release system can be placed in proximity of the prophylacticor therapeutic target, thus requiring only a fraction of the systemicdose (see. e.g., Goodson, In Medical Applications of Controlled Release,(1984), pages 115-138).

Controlled release systems are discussed in the review by Langer(Science, 249: 1527-1533 (1990)). Any technique known to one of skill inthe art can be used to produce sustained release formulations comprisingone or more therapeutic agents of the invention. See, e.g., U.S. Pat.No. 4,526,938; PCT Publication Nos. WO 91/05548 and WO 96/20698: Ning etal., “Intratumoral Radioimmunotherapy of a Human Colon Cancer XenograftUsing a Sustained-Release Gel,” Radiother. Oncol., 39: 179-189 (1996);Song et al., “Antibody Mediated Lung Targeting of Long-CirculatingEmulsions.” PDA J. Pharm. Sci. Tech., 50: 372-377 (1996); Cleek et al.,“Biodegradable Polymeric Carriers for a bFGF Antibody for CardiovascularApplication,” Proceed Intl. Symp. Control. Rel. Bioact. Mater., 24:853-854 (1997), and Lam et al., “Microencapsulation of RecombinantHumanized Monoclonal Antibody for Local Delivery,” Proceed. Intl. Symp.Control Rel. Bioact. Mater.: 24: 759-760 (1997), each of which isincorporated herein by reference in their entireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in viva to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular. e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun: Biolistic. Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., Proc. Natl. Acad. Sci. USA,88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral (e.g.,intravenous), intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If compositions of the invention are to be administered topically, thecompositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms. 19th ed., (Mack Publishing Co., Easton,Pa., 1995). For non-sprayable topical dosage forms, viscous tosemi-solid or solid forms comprising a carrier or one or more excipientscompatible with topical application and having a dynamic viscositypreferably greater than water are typically employed. Suitableformulations include, without limitation, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like,which are, if desired, sterilized or mixed with auxiliary agents (e.g.,preservatives, stabilizers, wetting agents, buffers, or salts) forinfluencing various properties, such as, for example, osmotic pressure.Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, preferably in combinationwith a solid or liquid inert carrier, is packaged in a mixture with apressurized volatile (e.g., a gaseous propellant, such as FREON®) or ina squeeze bottle. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms if desired. Examples ofsuch additional ingredients are well known in the art.

If a method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If a method of the invention comprises oral administration, compositionscan be formulated orally in the form of tablets, capsules, cachets,gelcaps, solutions, suspensions, and the like. Tablets or capsules canbe prepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose, or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc, or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art. Liquid preparations for oral administration maytake the form of, but not limited to, solutions, syrups or suspensions,or they may be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives, or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

A method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See. e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540 and 4,880,078; andPCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013. WO 98/31346,and WO 99/66903, each of which is incorporated herein by reference theirentireties. In a specific embodiment, an antibody of the invention,combination therapy, and/or composition of the invention is administeredusing Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass., US).

A method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

A method of the invention may additionally comprise administration ofcompositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

Methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, acomposition can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents or pharmaceutical compositions of theinvention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C., and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions of the invention is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically scaled containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml, or at least 1(1) mg/ml. The liquid form should be storedat between 2° C., and 8° C. in its original container.

The binding proteins of the invention can be incorporated into apharmaceutical composition suitable for parenteral administration.Preferably, the binding protein will be prepared as an injectablesolution containing 0.1-250 mg/ml antibody. The injectable solution canbe composed of either a liquid or lyophilized dosage form in a flint oramber vial, ampoule or pre-filled syringe. The buffer can be L-histidine(1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Othersuitable buffers include but are not limited to, sodium succinate,sodium citrate, sodium phosphate or potassium phosphate. Sodium chloridecan be used to modify the toxicity of the solution at a concentration of0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectantscan be included for a lyophilized dosage form, principally 0-10% sucrose(optimally 0.5-1.0%). Other suitable cryoprotectants include trehaloseand lactose. Bulking agents can be included for a lyophilized dosageform, principally 1-10% mannitol (optimally 2-4%). Stabilizers can beused in both liquid and lyophilized dosage forms, principally 1-50 mML-methionine (optimally 5-10 mM). Other suitable bulking agents includeglycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally0.005-0.01%). Additional surfactants include but are not limited topolysorbate 20 and BRIJ surfactants.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, a DLL4 binding protein described herein is administered byintravenous infusion or injection. In another preferred embodiment, aDLL4 binding protein is administered by intramuscular or subcutaneousinjection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The DLL4 binding proteins of the present invention can be administeredby a variety of methods known in the art, although for many therapeuticapplications, the preferred route/mode of administration is subcutaneousinjection, intravenous injection, or infusion. As will be appreciated bythe skilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R Robinson, ed., (Marcel Dekker, Inc., New York, 1978).

In certain embodiments, a binding protein of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a binding protein of the inventionis coformulated with and/or coadministered with one or more additionaltherapeutic agents that are useful for treating disorders in which DLL4activity is detrimental. For example, an anti-huDLL4 antibody orantibody portion of the invention may be coformulated and/orcoadministered with one or more additional antibodies that bind othertargets (e.g., antibodies that bind other cytokines or that bind cellsurface molecules). Furthermore, one or more binding proteins of theinvention may be used in combination with two or more of the foregoingtherapeutic agents. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies.

In certain embodiments, a DLL4 binding protein of the invention islinked to a half-life extending vehicle known in the art. Such vehiclesinclude, but are not limited to, the Fc domain, polyethylene glycol, anddextran. Such vehicles are described. e.g., in U.S. Pat. No. 6,660,843B1 and published PCT Publication No. WO 99/25044, which are herebyincorporated by reference.

In a specific embodiment, nucleic acid sequences comprising nucleotidesequences encoding a binding protein of the invention or anotherprophylactic or therapeutic agent of the invention are administered totreat, prevent, manage, or ameliorate a disorder or one or more symptomsthereof by way of gene therapy. Gene therapy refers to therapy performedby the administration to a subject of an expressed or expressiblenucleic acid. In this embodiment of the invention, the nucleic acidsproduce their encoded binding protein or prophylactic or therapeuticagent of the invention that mediates a prophylactic or therapeuticeffect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., Clin. Pharmacy, 12: 488-505(1993); Wu and Wu, Biotherapy, 3: 87-95 (1991); Tolstoshev, Ann. Rev.Pharmacol. Toxicol., 32: 573-596 (1993); Mulligan, Science, 260: 926-932(1993); and Morgan and Anderson, Ann. Rev. Biochem., 62: 191-217 (1993);Robinson, C., Trends Biotechnol., 11(5): 155 (1993). Methods commonlyknown in the art of recombinant DNA technology which can be used aredescribed in Ausubel et al. (eds.), Current Protocols in MolecularBiology (John Wiley & Sons, New York, 1993); and Kriegler, Gene Transferand Expression, A Laboratory Manual, (Stockton Press. New York, 1990).Detailed descriptions of various methods of gene therapy are disclosedin US Patent Application Publication No. 20050042664 A1, which isincorporated herein by reference.

In another aspect this invention provides a method of treating (e.g.curing, suppressing, ameliorating, delaying, or preventing the onset of,or preventing recurrence or relapse of) or preventing a DLL4-associatedtumor in a subject. The method includes administering to a subject aDLL4 binding protein, e.g., an anti-DLL4 antibody or fragment thereof asdescribed herein, in an amount sufficient to treat or prevent theDLL-associated tumor or cancer. The DLL4 antagonist, i.e., the anti-DLL4antibody or fragment thereof, may be administered to a subject alone orin combination with other therapeutic modalities as described herein.

DLL4 plays a critical role in the pathology associated with a variety ofdiseases involving immune and inflammatory elements, in particularcancer and tumor angiogenesis. Examples of DLL4-associated disordersinclude, but are not limited to, those disorders that adversely effectthe following biological processes: neuronal function and development;stabilization of arterial endothelial fate and angiogenesis; regulationof crucial cell communication events between endocardium and myocardiumduring both the formation of the valve primordial and ventriculardevelopment and differentiation; cardiac valve homeostasis, as well asimplications in other human disorders involving the cardiovascularsystem: timely cell lineage specification of both endocrine and exocrinepancreas; influencing of binary fate decisions of cells that must choosebetween the secretory and absorptive lineages in the gut; expansion ofthe hematopoietic stem cell compartment during bone development andparticipation in commitment to the osteoblastic lineage such asosteoporosis; regulation of cell-fate decision in mammary glands atseveral distinct development stages; and certain non-nuclear mechanisms,such as control of the actin cytoskeleton through the tyrosine kinaseAbl. More specifically, DLL4-associated disorders include, but are notlimited to, cancers, T-ALL (T-cell acute lymphoblastic leukemia),CADASIL (Cerebral Autosomal Dominant Arteriopathy with Sub-corticalInfarcts and Leukoencephalopathy), MS (Multiple Sclerosis), Tetralogy ofFallot, and Alagille syndrome. Preferably, antibodies andantigen-binding portions thereof as described herein are used to treatcancers and tumors.

Binding proteins according to the invention can be used alone or incombination, i.e., more than one DLL4-binding protein described herein,to treat a cancer, a tumor, or other disorder in which binding to,inhibition of, and/or neutralization of DLL4 is considered desirable orotherwise beneficial to the health of an individual.

It should be understood that DLL4 binding proteins of the invention canalso be used alone or in combination with an additional agent, e.g., atherapeutic agent, said additional agent being selected by the skilledpractitioner for its intended purpose. For example, the additional agentcan be a therapeutic agent that is recognized in the art as being usefulto treat a cancer, tumor, or other disease or condition in which bindingto or inhibition of DLL4 is considered to be desirable or advantageousfor treating the cancer, tumor, or other disease or condition. Theadditional agent also can be an agent that imparts a beneficialattribute to the therapeutic composition e.g., an agent which affectsthe viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents, if the combination is such that the formedcomposition can perform its intended function.

Preferred combinations are non-steroidal anti-inflammatory drug(s) alsoreferred to as NSAIDS, which include drugs like ibuprofen. Otherpreferred combinations are corticosteroids including prednisolone; thewell known side-effects of steroid use can be reduced or even eliminatedby tapering the steroid dose required when treating patients incombination with the anti-DLL4 antibodies of this invention.Non-limiting examples of therapeutic agents for rheumatoid arthritiswith which an antibody, or antibody portion, of the invention can becombined include the following: cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, interferons, EMAP-II,GM-CSF, FGF, and PDGF. Antibodies of the invention, or antigen bindingportions thereof, can be combined with antibodies to cell surfacemolecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45,CD69, CD80 (B7,1), CD86 (B7,2), CD90, CTLA or their ligands includingCD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the pro-tumorigenic or pro-angiogenic signaling pathways.Preferred examples therapeutic agents useful in the methods andcompositions of the invention include antineoplastic agents,radiotherapy, and chemotherapy such as DNA alkylating agents, cisplatin,carboplatin, anti-tubulin agents, paclitaxel, docetaxel, taxol,doxorubicin, gemcitabine, gemzar, anthracyclines, adriamycin,topoisomerase I inhibitors, topoisomerase II inhibitors, 5-fluorouracil(5-FU), leucovorin, irinotecan, receptor tyrosine kinase inhibitors(e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g., celecoxib), andkinase inhibitors.

The DLL4 binding proteins of the invention may also be combined withagents, such as methotrexate, 6-MP, azathioprine sulphasalazine,mesalazine, olsalazine chloroquinine/hydroxychloroquine, pencillamine,aurothiomalate (intramuscular and oral), azathioprine, colchicine,corticosteroids (oral, inhaled and local injection), beta-2adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines(theophylline, aminophylline), cromoglycate, nedocromil, ketotifen,ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolatemofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroidssuch as prednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signaling by proinflammatory cytokines such as orIL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors). IL-1b□TNFαconverting enzyme (TACE) inhibitors, T-cell□converting enzymeinhibitors, TNFα signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG(Lenercept)), SIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines(e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid,hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen,valdecoxib, sulfasalazine, methylprednisolone, meloxicam,methylprednisolone acetate, gold sodium thiomalate, aspirin,triamcinolone acetonide, propoxyphene napsylate/apap, folate,nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium,oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenacsodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl,salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen,alendronate sodium, prednisolone, morphine sulfate, lidocainehydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptylineHCl, sulfadiazine, oxycodone HCL/acetaminophen, olopatadine hcl,misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab,IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796,SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, andMesopram. Preferred combinations include methotrexate or leflunomide andin moderate or severe rheumatoid arthritis cases, cyclosporine.

Non-limiting examples of therapeutic agents for cancers with which aDLL4 binding protein of the invention can be combined include thefollowing: budenoside: epidermal growth factor, corticosteroids;cyclosporin; sulfasalazine; aminosalicylates; 6-mercaptopurine;azathioprine; metronidazole; lipoxygenase inhibitors; mesalamine;olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1receptor antagonists: anti-IL-1β monoclonal antibodies; anti-IL-6monoclonal antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; and antibodies to or antagonists of otherhuman cytokines or growth factors, for example, TNF, LT, IL-1, IL-2,IL-6, IL-7, IL-8, IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, andPDGF. Antibodies of the invention, or antigen binding portions thereof,can be combined with antibodies to cell surface molecules such as CD2,CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90, or theirligands. The antibodies of the invention, or antigen binding portionsthereof, may also be combined with agents, such as methotrexate,cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide.NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone,phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents,complement inhibitors, adrenergic agents, agents which interfere with orIL-1 (e.g., IRAK, NIK, signaling by proinflammatory cytokines such asTNFα IKK, p38 or MAP kinase inhibitors), IL-1β converting enzymeinhibitors. TNFα converting enzyme inhibitors. T-cell sig□nallinginhibitors such as kinase inhibitors, metalloproteinase inhibitors,sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin convertingenzyme inhibitors, soluble cytokine receptors and derivatives thereof(e.g., soluble p55 or p75 TNF receptors, SIL-1RI, SIL-1RII, SIL-6R), andanti-inflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13, and TGFβ).

Other examples of therapeutic agents with which a DLL4 binding proteinof the invention can be combined include the following: TNF antagonists,for example, anti-TNF antibodies, D2E7 (PCT Publication No. WO 97/29131:HUMIRA®), CA2 (REMICADE®), CDP 571, TNFR-Ig constructs, (p75TNFRIgG(ENBREL®) and p55TNFRIgG (LENERCEPT)) and PDE4 inhibitors. Bindingproteins of the invention can be combined with corticosteroids, forexample, budenoside and dexamethasone. Binding proteins of the inventionmay also be combined with agents such as sulfasalazine, 5-aminosalicylicacid, and olsalazine, and agents which interfere with synthesis oraction of proinflammatory cytokines such as IL-1, for example, IL-1βconverting enzyme inhibitors and IL-1ra. DLL4 binding proteins of theinvention may also be used with T cell signaling inhibitors, forexample, tyrosine kinase inhibitors 6-mercaptopurines. DLL4 bindingproteins of the invention can be combined with IL-11. Binding proteinsof the invention can be combined with mesalamine, prednisone,azathioprine, mercaptopurine, infliximab, methylprednisolone sodiumsuccinate, diphenoxylate/atrop sulfate, loperamide hydrochloride,methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water,hydrocodone bitartrate/apap, tetracycline hydrochloride, fluocinonide,metronidazole, thimerosal/boric acid, cholestyramine/sucrose,ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidinehydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen,promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin. folic acid,levofloxacin, methylprednisolone, natalizumab, and interferon-gamma.

Non-limiting examples of therapeutic agents with which a binding proteinof the invention can be combined include the following: aspirin,nitroglycerin, isosorbide mononitrate, metoprolol succinate, atenolol,metoprolol tartrate, amlodipine besylate, diltiazem hydrochloride,isosorbide dinitrate, clopidogrel bisulfate, nifedipine, atorvastatincalcium, potassium chloride, furosemide, simvastatin, verapamil hcl,digoxin, propranolol hydrochloride, carvedilol, lisinopril,spironolactone, hydrochlorothiazide, enalapril maleate, nadolol,ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalolhydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium,lisinopril/hydrochlorothiazide, felodipine, captopril, and bisoprololfumarate.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a binding protein of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the binding protein may bedetermined by a person skilled in the art and may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the binding protein to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the binding protein areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a DLL4 binding protein of theinvention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1: In Vitro Assays Used to Determine the FunctionalActivity of DLL4 Antibodies Example 1.1: Affinity Determination UsingBIACORE® Surface Plasmon Resonance Technology

The BIACORE® surface plasmon resonance assay (Biacore, Inc., Piscataway,N.J., US) determines the affinity of antibodies with kineticmeasurements of on-rate and off-rate constants. Binding of DLL4antibodies to a purified recombinant DLL4 extracellular domain isdetermined by surface plasmon resonance-based measurements with aBiacore® instrument (either a Biacore 2000, Biacore 3000, or BiacoreT100, GE Healthcare, Piscataway, N.J. US) using running buffer HBS-EPB(10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.1 mg/ml BSA and 0.005%surfactant P20) at 25° C. For example, approximately 9000 RU of goatanti-human Fc specific polyclonal antibody (Thermo Fisher ScientificInc., Rockford, Ill., US) diluted in 10 mM sodium acetate (pH 4.5) isdirectly immobilized across a CM5 research grade biosensor chip using astandard amine coupling kit according to manufacturer's instructions andprocedures at 25 μg/ml. Unreacted moieties on the biosensor surface areblocked with ethanolamine. For kinetic analysis, rate equations derivedfrom the 1:1 Langmuir binding model are fitted simultaneously tomultiple antigen injections (using global fit analysis) with the use ofScrubber 2 (BioLogic Software). Biacore Biaevaluation 4.0.1 software orBiacore T1(00 Evaluation software. Purified antibodies are diluted inrunning buffer for capture across goat anti-human Fc reaction surfaces.Antibodies to be captured as a ligand (1 μg/ml) are injected overreaction matrices at a flow rate of 10 μl/min. During the assay, allmeasurements were referenced against the capture surface alone (i.e.,with no captured anti-DLL4 antibody). The association and dissociationrate constants, K_(on) (M⁻¹s⁻¹) and K_(off) (s⁻¹) are determined under acontinuous flow rate of 80 μl/min. Rate constants are derived by makingkinetic binding measurements at different antigen concentrations rangingfrom 1.23-900 nM, as a 3-fold dilution series, and included buffer-onlyinjections (to be used for double referencing). The equilibriumdissociation constant K_(D) (M) of the reaction between antibodies andthe target antigen is then calculated from the kinetic rate constants bythe following formula: K_(D)=K_(off)/K_(on). Binding is recorded as afunction of time and kinetic rate constants are calculated. In thisassay, on-rates as fast as 10⁶ M⁻¹s⁻¹ and off-rates as slow as 10⁻⁶ s⁻¹can be measured.

Example 1.2: Binding of DLL4 Antibodies to Soluble DLL4 ExtracellularDomain as Determined by ELISA Method 1 (Capture ELISA).

96-well Nunc-Immuno plates (#439454) were coated with 5 μg/ml antibodyagainst human IgG (Fcg fragment specific. Jackson ImmunoResearch.#109-005-098, 100 μl/well) in D-PBS (Gibco #14190) and incubatedovernight at 4° C. ELISA plates were washed 3 times with wash buffer(PBS, 0.05% Tween-20) and then blocked with 200 ml/well blocking buffer(D-PBS, 1% BSA, 1 mM CaCl₂, 0.05% Tween-20) for 1 hour at 25° C. Plateswere washed 3 times and incubated with 100 μl/well DLL4 antibodies(0.0001-100 nM, 10-fold serial dilution in blocking buffer) for 1 hourat 25° C., and then washed again 3 times. Plates containing capturedDLL4 antibody were incubated with biotin-labeled human DLL4extracellular domain (10 nM in blocking buffer, 100 μl/well) for 1 hourat 25° C., washed 3 times, and incubated with streptavidin conjugatedwith HRP (KPL #474-3000, 1:10,000 dilution in blocking buffer, 100μl/well) for 1 hour at 25° C. After the final wash, plates wereincubated with 100 l/well ELISA substrate (1-Step Ultra TMB-ELISA.Pierce #340280). The reaction was stopped after 2 minutes at 25° C. with100 μl/well 2 N H2SO₄ and the absorbance was read at 450 nm. Data wereanalyzed using Graphpad Prism software and EC₅₀ values were reported.

Method 2 (Copper Coated Plate).

96-well copper-coated plates (Thermo Scientific #15143) were washed 3times with wash buffer (PBS, 0.05% Tween-20) before use and thenincubated with 100 μl/well of human DLL4-his or mouse DLL4-his or cynoDLL4-his at 1 μg/ml in PBS, 1 hour at 25° C. with shaking. Plates werethen washed 3 times, 100 μl/well of recombinant rat/human chimeric orrecombinant human anti-DLL4 antibodies were then added to the plate(0.00164-27 nM, 4-fold serial dilution in ELISA buffer=PBST, 10%Superblock (Pierce #37515)) for 1 hour at 25° C. with shaking and thenwashed again 3 times. Plates were incubated with goat anti-human HRP(Pierce #31412) (1:40,000 dilution in ELISA buffer, 100 μl/well) for 1hour at 25° C. with shaking, then washed 3 times. After the final wash,plates were incubated with 100 μl/well ELISA substrate (Sigma # T8665).The reaction was stopped after 8 minutes at 25° C. with 100 μl/well 1NHCl and the absorbance was read at 450 nm. Data were analyzed usingGraphpad Prism software and EC50 values were reported.

Example 1.3: Binding of DLL4 Monoclonal Antibodies to the Surface ofHuman Tumor Cell Lines as Assessed by Flow Cytometry (FACS)

Stable cell lines overexpressing cell-surface DLL4 were harvested fromtissue culture flasks, washed four times and resuspended in phosphatebuffered saline (PBS) containing 1% bovine serum albumin and 1 mM CaCl₂(FACS buffer), 1.5×10³ cells were incubated with antibodies at variousconcentrations in FACS buffer for 60 minutes on ice. Cells were washedtwice and 50 μL of R-phycoerythrin-conjugated anti-rat IgG. F(ab′)₂fragment (1:200 dilution in FACS buffer) (Jackson ImmunoResearch, WestGrove, Pa. US, Cat. #112-116-072) was added. Following an incubation onice (4° C., 60 minutes), cells were washed three times and resuspendedin FACS buffer. Fluorescence was measured using a Becton DickinsonFACSCalibur-HTS (Becton Dickinson, San Jose. Calif., US). Data wereanalyzed using Graphpad Prism software and EC₅₀ values were reported asthe concentration of antibody to achieve 50% of maximal DLL4 antibodiesbinding to DLL4 expressing cells.

Example 1.4: Inhibition of Notch-1 Interaction with Soluble DLL4Extracellular Domain by DLL4 Antibodies (Competition ELISA)

96-well Nunc-Immuno plates (#439454 for huDLL4 ELISA) and 96-well Costarplates (#9018 for muDLL4 ELISA) were coated with 16 nM human Notch-1(R&D Systems #3647-TK, 100 μl/well in D-PBS) and incubated overnight at4° C. Plates were then washed 3 times with wash buffer (PBS, 0.05%Tween-20) and blocked with 200 μl/well blocking buffer (D-PBS, 1% BSA, 1mM CaCl₂, 0.05% Tween-20) for 1 hour at 25° C. While blocking, biotinlabeled human DLL4 extracellular domain (14 nM) was mixed with antibody(30 pM-66 nM, 3-fold serial dilution in blocking buffer) for 1 hour at25° C. with shaking. Assay plates were washed after blocking, andincubated with DLL4/antibody mixtures (100 μl/well, 1 hour at 25° C.with shaking). Plates were washed again and 100 μl/well streptavidinconjugated with HRP (Fitzgerald #65R-S 104PHRPx, diluted 1:5,000 inblocking buffer) was added for 1 hour at 25° C. with shaking. After afinal wash, plates were developed using 100 μl/well substrate (TMB Sigma# T8665), and the reaction was stopped after an 8-minute incubation at25° C. (for muDLL4 ELISA) and after 20 minute incubation at 25° C. (forhuDLL4 ELISA) using 100 μl/well 1N HCl, and the absorbance was read at450 nm. Data were analyzed using Graphpad Prism software and IC₅₀ valueswere reported as the concentration of antibody to achieve 50% reductionof DLL4 bound to Notch1.

Example 1.5: Blocking of Soluble Notch Binding to DLL4-Overexpressing293G Cells by Anti-DLL4 Monoclonal Antibodies as Assessed by FlowCytometry (Competition FACS)

Notch blocking assay: Briefly, stable cell lines overexpressingcell-surface DLL4 were harvested from tissue culture flasks andre-suspended in phosphate buffered saline (PBS) containing 1% bovineserum albumin and 1 mM CaCl₂ (FACS buffer). HEK293-hDLL4 or HEK293-mDLL4cells were dispensed into 96-well plate (v-bottom) at 1.5×10⁵ cells/wellin FACS buffer. After spinning down cells and discarding thesupernatant, 50 μL of purified IgG with appropriate dilution was addedto each well, and incubated on ice at 4° C. for 60 minutes, followed byaddition of 50 μL/well of Notch1-biotin at 0.2 pg/mL for hDLL4-293G or2.0 μg/mL for mDLL4-293G (1.0 or 0.1 j±g/mL final) for additional 1 hourincubation ice at 4° C. After washing the cells two times with FACSbuffer, 50 μL of R-phycoerythrin-conjugated streptavidin (1:150 dilutionin FACS buffer) (Jackson ImmunoResearch, West Grove, Pa., US, catalogno. 016-110-084) were added. Following an incubation on ice (4° C., 60minutes), cells were washed three times and resuspended in FACS buffer.Fluorescence was measured using a Becton Dickinson FACSCalibur-HTS(Becton Dickinson, San Jose. Calif.). Data were analyzed using GraphpadPrism software and IC₅₀ values were reported as the concentration ofantibody to achieve 50% reduction of Notch1 bound to DLL4 expressingcells.

Example 1.6: Inhibition of DLL4-Dependent Increase of sVEGFR1 (sFLT1) inEA.Hy926 Cells by DLL4 Antibodies

Tissue culture plates, 96-well, were coated with 100 μl/well human DLL4extracellular domain at 1.67 mg/ml in D-PBS (Gibco #14190) and incubatedovernight at 4° C. Plates were washed once with D-PBS and 4000 EA.hy926cells/well were seeded in the absence or presence of antibodies. Cellproliferation was measured four days later using the CyQUANT CellProliferation Assay Kit (Invitrogen, # C35007), sVEGFR1 expression inthe conditioned media was detected by an ELISA kit per themanufacturer's recommendations (R&D Systems # DVR100B). Levels ofsVEGFR1 were normalized to the RFU determined by CyQUANT assay toaccount for differences in cell proliferation.

Example 1.7: Inhibition of DLL4-Dependent Notch Activation in EA.Hy926Cells by DLL4 Antibodies Using Notch Reporter Assay

96-well black clear-bottom tissue culture plates were seeded overnightwith 7000 cells/well engineered EA.hy926 cells expressing luciferasedriven by a Notch-responsive promoter. Antibodies serially diluted from200 nM were mixed for 15 minutes with equal volume of 5000 HEK293Gcells/well expressing full-length DLL4. The 293G/DLL4 cells wereco-cultured with EA.hy926 Notch reporter cells for 24 hrs in thepresence of testing antibodies. Luciferase activity was analyzed byPromega's substrate (Promega # E2940).

Example 1.8: Analytical Methods and Techniques for Molecule Identity andPhysicochemical Property Characterizations PEG Precipitation Method.

The use of PEG for inducing phase separation of a solid proteinaccording to principles of volume exclusion represents a feasibleapproach to assess the solubility of a protein. PEG has severaladvantages over other precipitants, including minimal denaturation ofproteins at ambient temperatures (does not affect tertiary structure ofproteins) and within the range of 4° C. to 30° C. temperature control isnot required, i.e., precipitation studies can be performed at ambienttemperature at the laboratory bench.

Generally, the precipitation of proteins by PEGs is explained on thebasis of volume exclusion effects. According to this theory, proteinsare sterically excluded from the regions of solvent that are occupied byPEG linear chains. As a result, proteins are concentrated and eventuallyprecipitated when their solubility is exceeded. In thermodynamic terms,the steric exclusion leads to an increase in the chemical potential ofthe protein until it exceeds that of the pure solid state, resulting inprotein precipitation. This happens mainly because of a largeunfavorable free energy of interaction between PEG and proteins,reducing the preferential hydration of protein due to steric exclusioneffects. In aqueous solutions, preferential hydration helps to maintainthe native structure of proteins. Generally, volume exclusion has beenshown to become more effective with increasing molecular weight of thePEG, i.e., less PEG is needed to precipitate proteins with increasingPEG molecular weight.

A PEG molecular weight of 3000 was chosen for estimating the solubilityof the antibodies covered by this patent. A 50% PEG solution was made bydissolving PEG in deionized water in the ratio of one gram of PEG to 1mL of water. The PEG solution is then added to a solution of antibodywhich is initially at a concentration of less than or equal to 0.5 mg/mland a volume of 0.5 mL. The PEG solution is continually added and mixeduntil the first instance of cloudiness persists. The percentage of PEG3000 needed to cause this precipitation is calculated as 50×(volume ofPEG 3000 solution added/initial volume of antibody solution before PEGaddition).

The percentage of PEG 3000 needed for precipitation is compared to thepercentage needed for precipitation of protein with known watersolubility. For example, the water solubility of adalimumab exceeds 200mg/mL. Consequently, if the percentage of PEG 3000 required toprecipitate a protein of interest is similar to the percentage needed toprecipitate adalimumab then the predicted solubility of that proteinwill be similar to the solubility adalimumab.

Real Solubility Method.

Real solubility is determined by using Amicon centrifugal filters toconcentrate a protein in solution until the protein is observed toprecipitate out of solution or until the minimum volume to which theprotein can be concentrated within the filter unit is reached. For thelatter, 15 mL Amicon centrifugal filters have a minimum volume ofapproximately 50 μl while 4 mL Amicon centrifugal filters have a minimumvolume of approximately 15 μl.

First a protein is dialyzed into a specific formulation(s). For thesestudies, the antibody amount was 10 mg or much less. Then the proteinsolution is inserted into the Amicon centrifugal filter retentatechamber. The chamber is lined with a nitrocellulose membrane with poresthat permit molecules of less than 10 to 30 kilodaltons to pass whensubjected to centrifugal force. Antibodies which are typically above 140kilodaltons will be retained while water, buffer molecules, smallexcipients, and salts will pass through. The centrifugal filter is thencentrifuged according to manufacturer specifications until the proteinis observed to precipitate out of solution or until the minimum volumeto which the protein can be concentrated within the filter unit isreached.

After centrifugation, the protein solution is removed from the retentatechamber and the concentration is measured by ultraviolet absorbance. Thesolution is then kept at 25° C., and 5° C. for 1 to 2 days and ismonitored for signs of precipitation.

Near UV-CD Technique.

Near UV-CD spectroscopy provides important information about thetertiary structure of proteins and is one of the most used techniques inthis regard. CD refers to the differential absorption of the left andright circularly polarized components of plane polarized radiation. Forproteins, the chromophores in the near UVCD region (250-320 nm) are thearomatic amino acids, i.e., tryptophan, tyrosine, and phenyl-alanine,and the disulfide bonds, and the CD effect occurs when the chromophoresare present in an asymmetric (buried) environment. Signals in the regionfrom 250-270 nm are attributable to phenylalanine residues, signals from270-290 nm are attributable to tyrosine, and those from 280-300 nm areattributable to tryptophan. Disulfide bonds give rise to broad weaksignals throughout the near-UV spectrum. The near-UV CD spectrum can besensitive to small changes in tertiary structure such as those due toprotein-protein interactions and/or changes in formulation conditions.

There are a number of other factors that can influence the CD spectra ofaromatic amino acids. Among these are: (1) the rigidity of the protein,(2) the nature of hydrogen bonding, and (3) interactions between variousaromatic amino acids. Additionally, proteins with large number of suchamino acids can have smaller CD bands due to the cancellation of thepositive and negative bands.

Briefly, a protein dialyzed into the desired formulation(s) at 1 mg/mland is scanned from 250-320 nm or 240-320 nm with a Jasco 800 CDspectrometer. The corresponding formulation without protein is alsoscanned and the readings subtracted from that of the scan of the proteinsolution. A near UV-CD spectra is a plot of molar ellipticities versuswavelength from 250 or 240 to 320 nm.

For antibodies in general, a near UV-CD spectrum with a semi-sigmoidalprofile indicates good tertiary structure folding while a flatter andless featured profile indicates a greater tendency to unfold. Compactfolding is associated with good stability while poor folding exposes thehydrophobic interior which may lead to hydrophobic interactions amongprotein molecules resulting in the formation of undesired aggregates.

DSC Technique.

The thermal stability of the antibodies was assessed using a DSCinstrument. The DSC instrument used was an automated VP-DSC equipmentwith Capillary Cell (Microcal, GE Healthcare Ltd./Microcal,Buckinghamshire, UK). Unfolding of molecules was studied applying a 1°C./minute scan rate over a 25° C.-95° C. temperature range for samplesat 1 mg/mL. Additional measurement parameters applied were a fittingperiod of 16 seconds, a pre-scan wait time of 10 minutes, andmeasurements were performed in none-feedback mode. Per individualmeasurement, 420 μL of sample/blank were filled into the DSC measurementsample holder, with a plate fill scheme as provided below. Thethermograms obtained were fitted to a non two state model to obtain themidpoint temperatures and enthalpies of the different transitions.

An additional requirement for successful biologics development candidateis that the protein remains its native state and conformation. A proteinin aqueous solution is in equilibrium between the native (folded)conformation and its denatured (unfolded) conformation. The stability ofthe native state is based on the magnitude of the Gibbs free energy (DG)of the system and the thermodynamic relationship between enthalpy (DH)and entropy (DS) changes. A positive DG indicates the native state ismore stable than the denatured state—the more positive the DG, thegreater the stability. For a protein to unfold, stabilizing forces needto be broken. Conformational entropy overcomes stabilizing forcesallowing the protein to unfold at temperatures where entropy becomesdominant. DSC measures DH of protein unfolding due to heat denaturation.As a general rule it can be stated that the higher the transitionmidpoint (the Tm), the more stable the protein at lower temperatures.During the same experiment DSC also measures the change in heat capacity(DCp) for protein denaturation. Heat capacity changes associated withprotein unfolding are primarily due to changes in hydration of sidechains that were buries in the native state, but become solvent exposedin the denatured state. DSC has been shown to be a valuable predictor ofliquid formulation stability for proteins and other biologicalmacromolecules (Remmele and Gombotz, BioPharm., 13: 36-46 (2000), andRemmele et al., Pharm. Res., 15: 200-208 (1998)).

SEC Technique.

Size exclusion chromatography was used to separate proteins based onsize. Proteins are carried in an aqueous mobile phase and through aporous stationary phase resin packed in a column. The retention time inthe column is a function of the hydrodynamic size of the protein and thesize of the pores in the packed resin bed. Smaller molecules canpenetrate into smaller pores in the resin and are retained longer thanlarger molecules. Upon elution from the column the proteins are detectedby UV absorbance. The SEC method used a TSK gel guard (TOSOHBiosciences, Montgomeryville, Pa. US, cat no. 08543) and a TSK gelG3000SWxL (TOSOH Biosciences, Montgomeryville. Pa., US, cat. no. 08541).The mobile phase was 100 mM Na₂HPO₄. 200 mM Na₂SO₄, pH 6.8. The flowrate was 0.25 mL/minute. Injection volume was 20 μL of 1 mg/mL sample.The column temperature was room temperature. The autosampler temperaturewas 2-8° C. The total run time was 55 minutes. The detection was basedon UV absorbance at 214 nm wavelength, with band width set at 8 nm,using reference wavelength at 360 nm with band width 100 nm.

Freeze-Thaw Method.

Antibody solutions at 1 mg % ml in the desired formulation(s) are frozenat −80° C. for at least 4 hours and are then thawed at 30° C. in a waterbath. The solution is then refrozen at −80° C. This is repeated for 5cycles. After certain freeze-thaw cycles, e.g., second and fourth, aportion of the solution may be withdrawn for analysis by SEC beforerefreezing. Freeze-thaw stability testing is done at low proteinconcentration in order obtain a “worse-case scenario” due to greaterexposure of protein molecules to the denaturing ice-water interfaces. Athigher concentrations, proportionally less protein encounters theice-water interface, instead interacting with other protein molecules.

Accelerated Stability Method.

Antibody solutions at 1 mg/ml in the desired formulation(s) are passedthrough 0.22 μm PVDF filters under sterile conditions and incubated at40° C., and/or 50° C. for at least 21 days. At 7 days and 21 days,aliquots are withdrawn under sterile conditions and subjected toanalysis by SEC. Solutions are then returned to incubation.

Example 2: Generation and Isolation of Anti-DLL4 Human MonoclonalAntibodies E9 and a 10 by PROfusion mRNA Display Technology

Using PROfusion mRNA display technology (see, Chung-Ming Hsieh et al.,US Patent Application Publication No. 2010/0099103), pooled human spleenand lymph node antibody libraries were selected seven rounds againstDLL4 antigens: 100 nM biotin-labeled human DLL4 extracellular domain(round 1 and 2), a mixture of 50 nM biotin-labeled human DLL4extracellular domain, and 50 nM biotin-labeled mouse DLL4 extracellulardomain (round 3), 100 nM biotin-labeled human DLL4 extracellular domain(round 4 and 5), 293G cells stably expressing human DLL4 and 100 nMbiotin-labeled human DLL4 extracellular domain (round 6), BAF3 cellsstably expressing human and mouse DLL4 (round 7). Both A10 and E9 wereidentified from the round 7 selection of the antibody libraries (Table4). When constructed in wild type human IgG1, they are renamed A10.1 andE9.1, respectively.

TABLE 4Anti-DLL4 PROfusion Fully Human Antibody Clones E9 and A10 SequenceInformation (Kabat numbered CDRs are indicated by underlining) V CloneSource Isotype Region Germline Sequence 12345678901234557890 E9 HumanhuIgG1: VH VH4-39 EVQLQESGPGLVKPSETLSL Lymph VL(λ), TCTVSGGSISSSSYYWGWIRNode VH(L234, QPPGKGLEWIGDIYYTGSTY 235A) YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARE DVILRGGSDYWGQGTLVTVS S (SEQ ID NO: 1) VL V2-1SYELTQPPSVSVSPGQTASI TCSGQRLGDKYASWYQQKPG QSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPM DEADYYCQAWDRDTGVFGYG TRVTVL (SEQ ID NO: 11) A10Human huIgG1: VH VH3-30 EVQLLESGGGLVKSGGSLRL Spleen VL(λ),SCAASGFTFRSHWMSWVRQA VH(L234, PGKGLEWVAIISYDGSNKYS 235A)ADSVKGRFTISRDNSKNTLY LQLNSLRAEDTAVYYCAKAG GGNVGFDIWGQGTMVTVSS(SEQ ID NO: 112) VL V2-1 LPVLTQPPSVSVSPGQTASI TCSADKLGTKYVSWYQQKPGQSPVLVIYQDAKRPSGIPER FSGSNSGNTATLTISGTQTM DEADYLCQSWDRSDVVFGGGTKVTVL (SEQ ID NO: 113)

Example 3: In Vitro Characterization of PROfusion Antibodies E9 and A10

The DLL4 antigen binding affinities for E9 and A10 were determined bythe BIACORE technology as described in Example 1.1. As shown in Table 5,below, E9 and A10 have similar equilibrium dissociation constant valuesagainst human DLL4 (K_(D) of 3.36 and 6.68 nM, respectively) andcynomolgus monkey DLL4 (K_(D) of 4.2 and 7.8 nM, respectively). E9 alsocross-reacts with mouse and rat DLL4 (K_(D) of 16 and 15 nM,respectively).

TABLE 5 Biacore Kinetics on Anti-DLL4 PROfusion Antibodies. Kinetics onBiacore huDLL4 ECD cynoDLL4 ECD muDLL4 ECD ratDLL4 ECD MAb Ka Kd K_(D)Ka Kd K_(D) Ka Kd K_(D) Ka Kd K_(D) E9 2.0 6.6 3.36 1.8 7.4 4.2 1.8 2.616 1.9 2.9 15 E+04 E−05 E+04 E−05 E+04 E−04 E+04 E−04 A10 2.2 1.5 6.681.9 1.5 7.8 — — NB — — NB E+04 E−04 E+04 E−04 MAb = monoclonal antibody;E = multiply by 10 to indicated exponent; Ka (M⁻¹s⁻¹); Kd (s⁻¹); K_(D)(nM); NB = nobinding (900 nM DLL4)

Antibody-antigen binding activity was also evaluated using ELISA andFACS based assays (described in Example 1.2, 1.3 and EC₅₀ valuesreported in Table 3). In addition to binding to recombinant DLL4extracellular domain (ECD). E9 and A10 can both bind DLL4 expressed atcell surface (Table 6).

The ability of the antibodies to block DLL4 interaction with itsreceptor Notch1 was assessed with ELISA and FACS based competitionassays as described in Example 1.4 and 1.5. As shown in Table 6, E9 andA10 efficiently blocked the interaction of Notch1 with DLL4 (ECD andcell-bound form). In addition, cell-based functional assays weredeveloped to further determine the ability of the antibodies toneutralize DLL4-mediated cellular activity in vitro (as described inExamples 1.6 and 1.7). Both E9 and A10 inhibited DLL4-induced Notchactivation and sVEGFR1 expression in EA.hy926 cells (Table 6).

TABLE 6 PROfusion DLL4 antibody in vitro potency. Functional BlockadeAssays Competition Competition sVEGFR1 Inhibition of Direct BindingAssays ELISA FACS Inhibition Notch Capture ELISA FACS (IC₅₀, nM) (IC₅₀,nM) (IC₅₀, nM) activation via (EC₅₀, nM) (EC₅₀, nM) DLL4 ECD/ huNotch-1/DLL4 ECD/ huDLL4 cells, DLL4 ECD DLL4 Cells huNotch-1 DLL4 cells EaHycells coculture MAb hu mu cyno hu mu hu mu cyno hu mu hu mu (IC₅₀, nM)E9 0.17 0.18 0.40 7.23 0.32 1.7 2.1 2.1 23.4 3.2 1.0 0.4 6.2 A10 0.17 —0.44 0.93 — 2.6 — 5.1  4.4 — 1.5 — 7.3 MAb = monoclonal antibody; hu =human; mu = murine; cyno = cynomolgus monkey

Example 4: Affinity Maturation of PROfusion Antibodies E9 and A10Anti-DLL4 E9 Affinity Maturation.

Sequence alignment showed that the DLL4 antibody E9 shares the highestidentity to human germlines VH4-39/JH4 and V2-1/JL6. To improve theaffinity of E9 to DLL4, hypermutated CDR residues were identified fromother human antibody sequences in the IgBLAST database that also sharedhigh identity to germlines VH4-39 and V2-1. The corresponding E9 CDRresidues were then subjected to limited mutagenesis by PCR with primershaving low degeneracy at these positions to create three antibodylibraries in the scFv format suitable for use an affinity maturationprocedure. The first library contained mutations at residues 30, 31, 32,33, 50, 54, 56, and 65 in the VH CDR1 and 2 (Kabat numbering); thesecond library at residues 95 to 100, 100a, 100b, 100c, and 102 in VHCDR3; and the third library at residues 27, 30, 31, 33, 52, 53, 93 to 96in the three VL CDRs. To further increase the identity of E9 to thehuman germline framework sequences, an Arg at VL position 103 wasmutated to a Lys and a binary degeneracy at VL positions 80 (A/P) and100 (S/Y) was also introduced into the third library (Table 7).

TABLE 7Mutations in E9 VH and VL Amino Acid Sequences for Affinity Maturation.Mutated E9 VH Sequence (SEQ ID NO: 114):EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYY                             NNGN        S           N N N                             G NT        L             A T                             R  G                      I G                                R                      S                                                       RNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS     N                      E         YAMAYGGAA D     D                                FSLPFS SL S     G                                QGPRMQ  V N                                      WV S A  R H                                      LE K      E                                      AN Q      R                                         V      L                                         G      P                                         M      C                                         E      I                                                MMutated E9 VL Sequence (SEQ ID NO: 115):SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPER                        ES  E  V                Q LT                        DG                        TE                         M                        AN                         K                        EQ                         L                        FS                         T                         MFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTRVTVL                  A            SEVA   S  K                               M AC                                E S                               N M                                K

These E9 libraries were transformed into cells and displayed on cellsurfaces to be selected against a low concentration of biotinylated DLL4extracellular domain by magnetic then fluorescence activated cellsorting (FACS). Selection for improved on-rate, off-rate, or both werecarried out and antibody protein sequences of affinity-modulated E9clones (Table 8) were recovered for converting back to IgG format forfurther characterization.

Table 8. Protein Sequences of Antibody Clones Identified from AffinityMaturation Libraries for Anti-DLL4 Antibody E9.

TABLE 8 Affinity Matured Clones: Heavy Chain (VH) Regions E9.4EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARYDVSLGGSSDHWGQGTLVTVSS (SEQ ID NO: 116) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSYDVSLGGSSDH (SEQ ID NO: 117) (SEQ ID NO: 118) (SEQ ID NO: 119) E9.11EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSPVTISVDTSKNQFSLKLSSVTAADTAVYYCAREAVPLGGGSDYWGQGTLVTVSS (SEQ ID NO: 120) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSEAVPLGGGSDY (SEQ ID NO: 121) (SEQ ID NO: 122) (SEQ ID NO: 123) E9.14EVQLQESGPGLVKPSETLSLTCTVSGGSISNSRYHWGWIRQSPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAEDTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 124) CDR1 CDR2 CDR3 NSRYHWG DIYYTGSTYYNPSLKSEDVILRGGSDY (SEQ ID NO: 125) (SEQ ID NO: 126) (SEQ ID NO: 127) E9.17EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGNGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREEAILGGGSDYWGQGTLVTVSS (SEQ ID NO: 128) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSEEAILGGGSDY (SEQ ID NO: 129) (SEQ ID NO: 130) (SEQ ID NO: 131) E9.18EVQLQESGPGLVKPSETLSLTCTVSGGSISSSGYYWGWIRQPPGKGLEWIGDINYAGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 132) CDR1 CDR2 CDR3 SSGYYWG DINYAGSTYYNPSLKSEDVILRGGSDY (SEQ ID NO: 133) (SEQ ID NO: 1134) (SEQ ID NO: 135) E9.19EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNTSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVSLGGGSDTWGQGTLVTVSS (SEQ ID NO:136) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSFDVSLCGGSDT (SEQ ID NO: 137) (SEQ ID NO: 138) (SEQ ID NO: 139) E9.22EVQLQESGPGLVKPSETLSLTCTVSGGSISNSRYHWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKGRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 140) CDR1 CDR2 CDR3 NSRYHWG DIYYTGSTYYNPSLKGEDVILRGGSDY (SEQ ID NO: 141) (SEQ ID NO: 142) (SEQ ID NO: 143) E9.48EVQLQESGPGLVKPSETLSLTCTVSGCSISSSGYYWGWIRQPPGKGLEWIGDINYRGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 144) CDR1 CDR2 CDR3 SSGYYWG DINYRGSTYYNPSLKSEDVILRGGSDY (SEQ ID NO: 145) (SEQ ID NO: 146) (SEQ ID NO: 147) E9.65EVQLQESGPGLVKPSETLSLTCTVSGGSIRNSRYHWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKGRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 148) CDR1 CDR2 CDR3 NSRYHWG DIYYTGSTYYNPSLKGEDVILRGGSDY (SEQ ID NO: 149) (SEQ ID NO: 150) (SEQ ID NO: 151) E9.66EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVPLGGGADKWGQGTLVTVSS (SEQ ID NO: 152) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSEGVPLCCGADK (SEQ ID NO: 153) (SEQ ID NO: 154) (SEQ ID NO: 155) E9.71EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQALAMGGGSDKWGQGTLVTVSS (SEQ ID NO: 156) CDR1 CDR2 CDR3 SSSYYMG DIYYTGSTYYNPSLKSQALAMGGGSDK (SEQ ID NO: 151) (SEQ ID NO: 158) (SEQ ID NO: 159) E9.13EVQLQESGPGLVKPSETLSLTCTVSGCSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 160) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSEDVILRGCSDY (SEQ ID NO: 161) (SEQ ID NO: 162) (SEQ ID NO: 163) E9.16EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 164) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSEDVILRGGSDY (SEQ ID NO: 165) (SEQ ID NO: 1166) (SEQ ID NO: 167) E9.38EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVILRGGSDYWGQGTLVTVSS (SEQ ID NO: 168) CDR1 CDR2 CDR3 SSSYYWG DIYYTGSTYYNPSLKSEDVILRGGSDY (SEQ ID NO: 169) (SEQ ID NO: 170) (SEQ ID NO: 171) E9.2BEVQLQESGPGLVKPSETLSLTCTVSGGSISSSNYYWGWIRQPPGKGLEWIGDINYNGNTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREAVALGGGADDWGQGTLVTVSS (SEQ ID NO: 172) CDR1 CDR2 CDR3 SSNYYWG DINYNGNTYYNPSLKSEAVALGGGADD (SED ID NO: 173) (SEQ ID NO: 174) (SEQ ID NO: 175) E9.1FEVQLQESGPGLVKPSETLSLTCTVSGGSISSGSYYWGWIRQPPGKGLEWIGDINYIGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREAVSFGGGADSWGQGTLVTVSS (SEQ ID NO: 176) CDR1 CDR2 CDR3 SGSYYWG DINYIGSTYYNPSLKSEAVSFGGGADS (SEQ ID NO: 177) (SEQ ID NO: 178) (SEQ ID NO: 179) E9.10HEVQLQESGPGLVKPSETLSLTCTVSGGSISSSGYYWGWIRQPPGKGLEWIGDIYYTGNTYYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTLVYYCAREEVILGGGADQWGQGTLVTVSS (SEQ ID NO: 180 CDR1 CDR2 CDR3 SSGYYWG DIYYTGNTYYNPSLKNEEVILGGGADQ (SEQ ID NO: 181) (SEQ ID NO: 182) (SEQ ID NO: 183) E9.5EEVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDINYIGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARESVPLGGGADEWGQGTLVTVSS (SEQ ID NO: 184) CDR1 CDR2 CDR3 SSSYYWG DINYIGSTYYNPSLKSESVPLGGGADE (SEQ ID NO: 185) (SEQ ID NO: 186) (SEQ ID NO: 187) E9.10CEVQLQESGPGLVKPSETLSLTCTVSGGSISSGSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTLVYYCARQAVMYGGGSDNWGQGTLVTVSS (SEQ ID NO: 188) CDR1 CDR2 CDR3 SGSYYWG DIYYTGSTYYNPSLKSQAVMYGGGSDN (SEQ ID NO: 189) (SEQ ID NO: 190) (SEQ ID NO: 191) E9.7EEVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYAGSTYYNPSLKDRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDMILGGGADNWGQGTLVTVSS (SEQ ID NO: 192) CDR1 CDR2 CDR3 SSSYYWG DIYYAGSTYYNPSLKDEDMILGGGADN (SEQ ID NO: 193) (SEQ ID NO: 194) (SEQ ID NO: 195) E9.12BEVQLQESGPGLVKPSETLSLTCTVSGGSISSSNYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREAVSFGGGADSWGQGTLVTVSS (SEQ ID NO: 196) CDR1 CDR2 CDR3 SSNYYWG DIYYTGSTYYNPSLKSEAVSFGGGADS (SEQ ID NO: 197) (SEQ ID NO: 198) (SEQ ID NO: 199) E9.10EEVQLQESGPGLVKPSETLSLTCTVSGGSINSGNYYWGWIRQPPGKGLEWIGDISYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVMYGGGGDSWGQGTLVTVSS (SEQ ID NO: 200) CDR1 CDR2 CDR3 SGNYYWG DISYTGSTYYNPSLKSEDVMYGGGGDS (SEQ ID NO: 201) (SEQ ID NO: 202) (SEQ ID NO: 203) E9.6AEVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDINYAGSTYYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREAVALGGGADSWGQGTLVTVSS (SEQ ID NO: 204) CDR1 CDR2 CDR3 SSSYYWG DINYAGSTYYNPSLKNEAVALGGGADS (SEQ ID NO: 205) (SEQ ID NO: 206) (SEQ ID NO: 207) E9.7AEVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDINYAGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVKFGGGADLWGQGTLVTVSS (SEQ ID NO: 208) CDR1 CDR2 CDR3 SSSYYWG DINYAGSTYYNPSLKSEDVKFGGGADL (SEQ ID NO: 209) (SEQ ID NO: 210) (SEQ ID NO: 211) E9.8HEVQLQESGPGLVKPSETLSLTCTVSGGSISSGSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARESVPLGGGADNWGQGTLVTVSS (SEQ ID NO: 212) CDR1 CDR2 CDR3 SGSYYWG DIYYTGSTYYNPSLKNESVPLGGGADN (SEQ ID NO: 213) (SEQ ID NO: 214) (SEQ ID NO: 215)Affinity Matured Clones: Light Chain (VL) Regions E9.13SYELTQPPSVSVSPGQTASITCSGDTLGDKYVSWYQUPGQSPVLVIYEDSERPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDSETGVEGSGTKVTVL (SEQ ID NO: 216)CDR1 CDR2 CDR3 SGDTLGDKYVS EDSERPS QAWDSETGV (SEQ ID NO: 217)(SEQ ID NO: 218) (SEQ ID NO: 219) E9.16SYELTQPPSVSVSPGQTASITCSGERLGDKYVSWYQQKPGQSPVLVIYEDFKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 220)CDR1 CDR2 CDR3 SGERLGDKYVS EDFKRPS QAWDRDTGV (SEQ ID NO: 221)(SEQ ID NO: 222) (SEQ ID NO: 223) E9.38SYELTQPPSVSVSPGQTASITCSGQRLGDKYVSWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDVGVFGSGTKVTVL (SEQ ID NO: 224)CDR1 CDR2 CDR3 SGQRLGDKYVS EDSKRPS QAWDRDVGV (SEQ ID NO: 225)(SEQ ID NO: 226) (SEQ ID NO: 221) E94:SYELTPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 228)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 229)(SEQ ID NO: 230) (SEQ ID NO: 231) E9.11SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 232)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 233)(SEQ ID NO: 234) (SEQ ID NO: 235) E9.14SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 236)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 237)(SEQ ID NO: 238) (SEQ ID NO: 239) E9.17SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 240)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 241)(SEQ ID NO: 242) (SEQ ID NO: 243) E9.18SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 244)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 245)(SEQ ID NO: 246) (SEQ ID NO: 247) E9.19SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 248)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 249)(SEQ ID NO: 250) (SEQ ID NO: 251) E9.22SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 252)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 253)(SEQ ID NO: 254) (SEQ ID NO: 255) E9.48SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 256)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 257)(SEQ ID NO: 258) (SEQ ID NO: 259) E9.65SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGYFGYGTKVTVL (SEQ ID NO: 260) CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV(SEQ ID NO: 261) (SEQ ID NO: 262) (SEQ ID NO: 263) E9.66SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 264)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 265)(SEQ ID NO: 266) (SEQ ID NO: 267) E9.71SYELTQPPSVSVSPGQTASITCSGQRLGDKYASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDRDTGVFGYGTKVTVL (SEQ ID NO: 268)CDR1 CDR2 CDR3 SGQRLGDKYAS EDSKRPS QAWDRDTGV (SEQ ID NO: 269)(SEQ ID NO: 210) (SEQ ID NO: 271) E9.2BSYELTQPPSVSVSPGQTASITCSGEGLGDKYVSWYQQKPGQSPVLVIYEDSTRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDSETGVFGSGTKVTVL (SEQ ID NO: 272)CDR1 CDR2 CDR3 SGEGLGDKYVS EDSTRPS QAWDSETGV (SEQ ID NO: 273)(SEQ ID NO: 274) (SEQ ID NO: 275) E9.1FSYELTQPPSVSVSPGQTASITCSGDRLGDKYVSWYQQKPGQSPVLVIYEDSQRPSGIPERFSGSNSGDTATLTISGTQAMDEADYYCQAWDMEAGVFGSGTKVTVL (SEQ ID NO: 276)CDR1 CDR2 CDR3 SGDRLGDKYVS EDSQRPS QAWDMEAGV (SEQ ID NO: 277)(SEQ ID NO: 278) (SEQ ID NO: 279) E9.10SYELTQPPSVSVSPGQTASITCSGDSLGDKYVSWYQQKPGQSPVLVIYEDSERPS CGIPERFSGSNSGDTATLTISGTQAMDEADYYCQAWDSETGVFGSGTKVT (SEQ ID NO: 280) CDR1CDR2 CDR3 SGDSLGDKYVS EDSERPS QAWDSETGV (SEQ ID NO: 281)(SEQ ID NO: 282) (SEQ ID NO: 283) E9.10SYELTQPPSVSVSPGQTASITCSGEGLGDKYVSWYQQKPGQSPVLVIYEDSERPS EGIPERFSGSNSGDTATLTISGTQAMDEADYYCQAWDSEAGVFGSGTKVT (SEQ ID NO: 284) CDR1CDR2 CDR3 SGEGLGDKYVS EDSERPS QAWDSEAGV (SEC ID NO: 285)(SEQ ID NO: 286) (SEQ ID NO: 287) E9.7ESYELTQPPSVSVSPGQTASITCSGDRLGDKYVSWYQQKPGQSPVLVIYEDSERPSGIPERFSGSNSGDTATLTISGTQAMDEADYYCQAWDSEAGVFGSG TKVT (SEQ ID NO: 288)CDR1 CDR2 CDR3 SGDRLGDKYVS EDSERPS QAWDSEAGV (SEQ ID NO: 289)(SEQ ID NO: 290) (SEQ ID NO: 291) E9.5ESYELTQPPSVSVSPGQTASITCSGDMLGDKYVSWYQQKPGQSPVLVIYEDSQRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDSETGVFGSGTKVT (SEQ ID NO: 292) CDR1CDR2 CDR3 SGDMLGDKYVS EDSQRPS QAWDSETGV (SEQ ID NO: 293)(SEQ ID NO: 294) (SEQ ID NO: 295) E9.12SYELTQPPSVSVSPGQTASITCSGDGLGDKYVSWYQQKPGQSPVLVIYEDSTRPS BGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDSESGVFGSGTKVT (SEQ ID NO: 296) CDR1CDR2 CDR3 SGDGLGDKYVS EDSTRPS QAWDSESGV (SEQ ID NO: 297)(SEQ ID NO: 298) (SEQ ID NO: 299) E9.10SYELTQPPSVSVSPGQTASITCSGESLGDKYVSWYQQKPGQSPVLVIYEDSKRPS HGIPERFSGSNSGDTATLTISGTQAMDEADYYCQAWDGETGVFGSGTKVT (SEQ ID NO: 300) CDR1CDR2 CDR3 SGESLGDKYVS EDSKRPS QAWDGETGV (SEQ ID NO: 301)(SEQ ID NO: 302) (SEQ ID NO: 303) E9.6ASYELTQPPSVSVSPGQTASITCSGDMLGDKYVSWYQQKPGQSPVLVIYEDTNRPSCIPERFSGSNSGDTATT,TISGTQAMDEADYYCQAWDSETGVEGSGTKVT (SEQ ID NO: 304) CDR1CDR2 CDR3 SGDMLGDKYVS EDTNRPS QAWDSETGV (SEQ ID NO: 305)(SEQ ID NO: 306) (SEQ ID NO: 307) E9.7ASYELTQPPSVSVSPGQTASITCSGESLGDKYVSWYQQKPGQSPVLNIYQDAMRPSGIPERFSGSNSGDTATLTISGTQAMDEADYYCQAWDMETGVFGSGTKVT (SEQ ID NO: 308) CDR1CDR2 CDR3 SGESLGDKYVS QDAMRPS QAWDMETGV (SEQ ID NO: 309)(SEQ ID NO: 310) (SEQ ID NO: 3111) E9.8HSYELTQPPSVSVSPGQTASITCSGESLGDKYVSWYQQKPGQSPVLVIYEDSMRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAWDSEVGVFGSGTKVT (SEQ ID NO: 312) CDR1CDR2 CDR3 SGESLGDKYVS EDSMRPS QAWDSEVGV (SEQ ID NO: 313)(SEQ ID NO: 314) (SEQ ID NO: 315)

Anti-DLL4 A10 Affinity Maturation.

In a manner similar to E9 affinity maturation described above, sequencealignment showed that the DLL4 antibody A10 shared the highest identityto human germlines VH3-30 and V2-1. Human VH and VX, sequences derivedfrom VH3-30 and V2-1, respectively, were downloaded from NCBI IgBlastdatabase to generate sequence logos. These sequence logos were used todecide which positions would be doped to generate the affinity maturedlibraries.

The A10 libraries were transformed into cells and displayed on the cellsurface to be selected against low concentration of biotinylated DLL4extracellular domain by magnetic then fluorescence activated cellsorting. Selection for improved on-rate or off-rate or both were carriedout and antibody protein sequences of affinity-modulated A10 clones wererecovered for converting back to IgG format for furthercharacterization. The heavy chain (VH) regions of the affinity maturedclones are shown below in Table 9 and the light chain (VL) regions ofthe affinity matured clones are shown below in Table 10.

TABLE 9Variable Heavy Chain Regions (VH) of Affinity Matured A10 Clones.Sequence Protein Region 123456789012345678901234567890 A10.3EVQLVESGGGLVKSGGSLRLSCAASGFTFR VH SHWMSWVRQAPGKGLEWVAIISYDGSNKYSADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCAKAGGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 316) A10.3 CDR-H1 Residues 31- SHWMS VH 35 of SEQ IDNO.: 315 A10.3 CDR-H2 Residues 50- IISYDGSNKYSADSVKG VH 66 of SEQ IDNO.: 316 A10.3 CDR-H3 Residues 99- AGGGNVGFDI VH 108 of SEQ ID NO.: 316A10.K30 EVQLVESGGGVVQPGGSLRLSCAASGFTFG VH NHWMSWVRQAPGKGLEWVADISSDGRYKYYADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCAKAGGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 317) A10.K30 CDR-H1 Residues 31- NHWMS VH 35 of SEQ IDNO.: 317 A10.K30 CDR-H2 Residues 50- DISSDGRYKYYADSVKG VH 66 of SEQ IDNO.: 317 A10.K30 CDR-H3 Residues 99- AGGGNVGFDI VH 108 off SEQ IDNO.: 317 A10.K42 EVQLVESGGGLVQPGGSLRLSCAASGFTFQ VHSHWMSWVRQAPGKGLEWVAMISYDGTIKYY ADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAKAGGGNVGFDIWGQGTMVTVSS (SEQ ID NO: 318) A10.K42 CDR-H1Residues 31- SHWMS VH 35 of SEQ ID NO.: 318 A10.K42 CDR-H2 Residues 50-MISYDGTIKYYADSVKG VH 66 of SEQ ID NO.: 318 A10.K42 CDR-H3 Residues 99-AGGGNVGFDI VH 108 of SEQ ID NO.: 318 A10.9AEVQLVESGGGVVQPGRSLRLSCAASGFTFR VH SYWMSWVRQAPGKGLEWVATISYDGRNKDYADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCAKAGGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 319) A10.9A Residues 31- SYWMS VH 35 of SEQ ID NO.: 319A10.9A Residues 50- TISYDGRNKDYADSVKG VH 66 of SEQ ID NO.: 319 A10.9AResidues 99- AGGGNVGFDI VH 108 of SEQ ID NO.: 319 A10.8AEVQLVESGGGVVQPGGSLRLSCAASGFTFG VH NHWMSWVRQAPGKGLEWVADISSDGRYKYYADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCAKAGGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 320) A10.8A Residues 31- NHWMS VH 35 of SEQ ID NO.: 320A10.8A Residues 50- DISSDGRYKYYADSVKG VH 66 of SEQ ID NO.: 320 A10.8AResidues 99- AGGGNVGFDI VH 108 of SEQ ID NO.: 320 A10.1AEVQLVESGGGVVQPGGSLRLSCAASGFTFH VH SHWMSWVRQAPGKGLEWVAMISDDGRNKDYADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCARAAGGNVGLDIWGQGTMVTVSS(SEQ ID NO: 321) A10.1A Residues 31- SHWMS VH 35 of SEQ ID NO.: 321A10.1A Residues 50- MISDDGRNKDYADSVKG VH 66 of SEQ ID NO.: 321 A10.1AResidues 99- AAGGNVGLDI VH 108 of SEQ ID NO.: 321 A10.5DEVQLVESCGGVVQSGGSLRLSCAASGFTFG VH SHWMSWVRQAPGKGLEWVADISVDGSNKYSADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAGGNVGLDSWGQGTMVTVSS(SEQ ID NO: 322) A10.5D Residues 31- SHWMS VH 35 of SEQ ID NO.: 322A10.5D Residues 50- DISVDGSNKYSADSVKG VH 66 of SEQ ID NO.: 322 A10.5DResidues 99- AAGGNVGLDS VH 108 of SEQ ID NO.: 322 A10.3AEVQLVESGGGVVQPGGSLRLSCAASGFTFG VH NHWMSWVRQAPGKGLEWVADISSDGRYKYYADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCAKAGGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 323) A10.3A Residues 31- NHWMS VH 35 of SEQ ID NO.: 32310.3A Residues 50- DISSDGRYKYYADSVKG VH 66 of SEQ ID NO.: 323 A10.3AResidues 99- AGGGNVGFDI VH 108 of SEQ ID NO.: 323 A10.6BEVQLVESGGGVVQPGGSLRLSCAASGFTFG VH NHWMSWVRQAPGKGLEWVADISSDGRYKYYADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCAKAGGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 324) A10.6B Residues 31- NHWMS VH 35 of SEQ ID NO.: 324A10.6B Residues 50- DISSDGRYKYYADSVKG VH 66 of SEQ ID NO.: 324 A10.6BResidues 99- AGGGNVGFDI VH 108 of SEQ ID NO.: 324 A10.3DEVQLVESGGGVVQPGRSLRLSCAASGFTFR VH SHWMSWVRQAPGKGLEWVADISQDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAPAAGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 325) A10.3D Residues 31- SHWMS VH 35 of SEQ ID NO.: 325A10.3D Residues 50- DISQDGSYKYYADSVKG VH 66 of SEQ ID NO.: 325 A10.3DResidues 99- AAGGNVGFDI VH 108 of SEQ ID NO.: 325 A10.4CEVQLVESGGGVVQPGGSLRLSCAASGFTFG VH SHWMSWVRQAPGKGLEWVADISNDGRYAYSADSVKGRFTISRDNSKNTLYLQLNSLRAED TAVYYCAKAGGGNVGFDIWGQGTMVTVSS(SEQ ID NO: 326) A10.4C Residues 31- SHWMS VH 35 of SEQ ID NO.: 326A10.4C Residues 50- DISNDGRYAYSADSVKG VH 66 of SEQ ID NO.: 326 A10.4CResidues 99  AGGGNVGFDI VH 108 of SEQ ID NO.: 326

TABLE 10Variable Light Chain Regions (VL) of Affinity Matured A10 Clones.Sequence Protein region 123456789012345678901234567890 A10.3SYELTQPPSVSVSPGQTASITCSADKLGTK VL YVSWYQQKPGQSPVLVIYQDAKRPSGIPERFSGSNSGNTATLTISGTQTMDEADYLCQSW DRSDVVFGGGTKLTVL (SEQ ID NO: 327) A10.3CDR-L1 Residues 23- SADKLGTKYVS VL 33 of SEQ ID NO.: 327 A10.3 CDR-L2Residues 49- QDAKRPS VL 55 of SEQ ID NO.: 327 A10.3 CDR-L3 Residues 88-QSWDRSDVV VL 96 of SEQ ID NO.: 327 A10.L45SYELTQPPSVSVSPGQTASITCSADELGTQ VL YVSWYQQKPGQSPVLVIYQDATRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAW DRSGVVFGGGTKLTVL (SEQ ID NO: 328) A10.L45CDR-L1 Residues 23- SADELGTQYVS VL 33 of SEQ ID NO.: 328 A10.L45 CDR-L2Residues 49- QDATRPS VL 55 of SEQ ID NO.: 328 A10.L45 CDR-L3Residues 88- QAWDRSGVV VL 96 of SEQ ID NO.: 328 A10.L73SYELTQPPSVSVSPGQTASITCSGDNLGSQ VL YVSWYQQKPGQSPVLVIYQDAQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAW DRSGVVFGGGTKLTVL (SEQ ID NO: 329) A10.L73CDR-L1 Residues 23- SGDNLGSQYVS VL 33 of SEQ ID NO.: 329 A10.L73 CDR-L2Residues 49- QDAQRPS VL 55 of SEQ ID NO.: 329 A10.L73 CDR-L3Residues 88- QAWDRSGVV VL 96 of SEQ ID NO.: 329 A10.3ASYELTQPPSVSVSPGQTASITCSADNLGEK VL YVSWYQQKPGQSPVLVIYQDATRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQSW DSSGAVFGGGTKLTVL (SEQ ID NO: 330) A10.3ACDR-L1 Residues 23- SADNLGEKYVS VL 33 of SEQ ID NO.: 330 A-10.3A CDR-L2Residues 49- QDATRPS VL 55 of SEQ ID NO.: 330 A10.3A CDR-L3 Residues 88-QSWDSSGAV VL 96 of SEQ ID NO.: 330 A10.6B SYELTQPPSVSVSPGQTASITCSADNLGNQVL YVSWYQQKPGQSPVLVIYQDGMRPSGIPER FSGSNSGNTATLTISGTQAMDEADYYCQAWDWSGEVFGGGTKLTVI (SEQ ID NO: 331) A10.6B CDR-L1 Residues 23- SADNLGNQYVSVL 33 of SEQ ID NO.: 331 A10.6B CDR-L2 Residues 49- QDGMRPS VL55 of SEQ ID NO.: 331 A10.6B CDR-L3 Residues 88- QAWDWSGEV VL96 of SEQ ID NO.: 331 A10.3D SYELTQPPSVSVSPGQTASITCSADKLGTK VLYVSWYQQKPGQSPVLVIYQDGNRPSGIPER FSGSNSGNTATLTISGTQAMDEADYYCQSWDQSGAVFGGGTKLTVL (SEQ ID NO: 332) A10.3D CDR-L1 Residues 23- SADKLGTKYVSVL 33 of SEQ ID NO.: 332 A10.3D CDR-L2 Residues 49- QDGNRPS VL35 of SEQ ID NO.: 332 A10.3D CDR-L3 Residues 88- QSWDQSGAV VL96 of SEQ ID NO.: 332 A10.4C SYELTQPPSVSVSPGQTASITCSADNLGNQ VLYVSWYQQKPGQSPVLVIYQDGMRPSGIPER FSGSNSGNTATLTISGTQAMDEADYYCQAWDSSGAVFGGGTKLTVL (SEQ ID NO: 333) A10.4C CDR-L1 Residues 23- SADNLGNQYVSVL 33 of SEQ ID NO.: 333 A10.4C CDR-L2 Residues 49- QDGMRPS VL55 of SEQ ID NO.: 333 A10.4C CDR-L3 Residues 88- QAWDSSGAV VL96 of SEQ ID NO.: 333

Example 5: Construction of CDR-Grafted E9 Antibodies

CDRs of E9 VH were grafted onto VH3 consensus framework (graftedVH=E9vh3g2) and CDRs of E9 VL were grafted onto VL2-1 framework ofanti-DLL4 A10 antibody (grafted VL=E9a 10vlg2) and onto VK framework ofA10 germline (the closest germline in homology to the E9 VL) (graftedVL=E9AVK). Alternatively framework (FW) repairing was also performedwith the E9 VH; its FW was maintained but amino acids that may causeantibody instability were replaced, (repaired VH=E9VH4r2). Frameworkback-mutations were incorporated in both CDR-grafting and FW-repairingto maintain antibody structure and functionality (Table 11 and Table14).

CDRs of E9 were also grafted on the frameworks of anti-IL-18 andanti-IL-12 antibodies. Specifically CDRs of E9 VH were grafted ontoVH5-51 framework of anti-IL-18 (grafted VH=E9VH325) and onto VH2-70framework of anti-IL-12 (grafted VH=E9VH1D4.1). CDRs of E9 VL weregrafted onto VK L2/L16 framework of anti-IL-12 (grafted VL=E9VL325) andonto VK B3 framework of anti-IL-12 (grafted VL=E9VL1D4.1). Frameworkback-mutations were incorporated in the CDR-grafting to maintainantibody structure and functionality (Table 15 and Table 18).

In silico constructed CDR grafted antibodies described above weresynthesized directly in the plasmid of choice by Blue HeronBiotechnology. The variable heavy region was inserted in-frame onto acDNA fragment encoding the wild type human IgG1 constant region and ontothe human IgG1 constant region containing two hinge-region amino acidmutations. These mutations are a leucine to alanine change at position234 (EU numbering) and a leucine to alanine change at position 235 (Lundet al., J. Immunol., 147: 2657 (1991)). The variable light chain regionwas inserted in-frame with the human lambda constant region and with thehuman kappa constant region. Upon receipt of synthesized constructs fromBlue Heron, DNA was scaled up and sequence confirmed. CorrectCDR-grafted heavy and light chains corresponding to each antibody(Tables 11 and 14) (Tables 15 and 18) were co-transfected intoHEK-293-6E cells to transiently produce full-length CDR-graftedanti-human DLL4 antibodies. Table 11 summarizes all the E9 antibodyvariants generated and HEK-293-6e expression data. Cell supernatantscontaining recombinant human antibody were purified by Protein ASepharose chromatography and bound antibody eluted by addition of acidbuffer. Antibodies were dialyzed into PBS.

The ability of purified CDR grafted antibodies to bind to DLL4 or toinhibit DLL4 activity was determined using different types of assayslike ELISA (Example 1.2, Method 2). Biacore (Example 1.2) and FlowCytometry (FACS) (Example 1.3). Table 12 and Table 16 show EC50 valuesfrom the ELISA assays and the FACS assays and the affinity determined byBiacore of the CDR grafted antibodies described respectively in Table 11and Table 15 for human DLL4, murine DLL4 and cynomolgus DLL4. Table 13and Table 17 show IC50 values from the blocking ELISA (Example 1.4) andblocking FACS (Example 1.5) with human DLL4 and murine DLL4 for theCDR-grafted antibodies described respectively in Table 11 and Table 15.

TABLE 11 Summary of the E9 antibody variants generated and expressiondata HEK-293-6e titer Antibody VH name VL name (mg/L) E9-SE1 E9vh3g2E9a10vlg2 104 E9-SE2 E9vh3g2 E9.1 87 E9-SE3 E9vh4r2 E9.1 89 E9-SE4 E9.1E9a10vlg2 66 E9-SE5 E9.1 E9AVK 79 E9-SE6 E9vh4r2 E9a10vlg2 125 E9-SE7E9vh3g2 E9AVK 56 E9-SE8 E9vh4r2 E9AVK 85

TABLE 12 Binding affinities of the E9 antibody variants for human,mouse, and cynomolgus DLL4 as determined by ELISA. Biacore, and FACS.Binding Data Human DLL4 Mouse DLL4 Cyno DLL4 Binding Binding BindingELISA Biacore Binding ELISA Biacore Binding ELISA Biacore (EC50, (Kd,FACS (EC50, (Kd, FACS (EC50, (Kd, Antibody nM) nM) (Kd, nM) nM) nM) (Kd,nM) nM) nM) E9-SE1 0.16 15.2* 3.45 0.19 12.83 1.34 0.14 5.42 E9-SE2 0.182.43 5.2 0.20 15.09 0.87 0.14 3.68 E9-SE3 0.18 2.26 2.29 0.20 12.1 0.740.14 3.42 E9-SE4 0.17 2.38 nc 0.20 10.52 1.15 0.14 3.92 E9-SE5 0.16 1.577.81 0.19 12.77 2.9 0.13 6.7 E9-SE6 0.17 0.64 4.25 0.19 10.04 1.18 0.136.55 E9-SE7 0.17 1.69 2.28 0.17 16.22 2.24 0.12 7.53 E9-SE8 0.18 2.002.67 0.19 13.03 2.32 0.14 3.41 E9.1 0.29 2.38 0.35 0.34 12.77 1.6* 0.244.08 *historic data; nc = not calculated

TABLE 13 Neutralizing activities of the E9 antibody variants for humanand mouse DLL4 as determined by ELISA and FACS. Functional Data HumanDLL4 Mouse DLL4 Blocking Blocking Blocking Blocking ELISA FACS ELISAFACS Antibody (IC50, nM) (IC50, nM) (IC50, nM) (IC50, nM) E9-SE1 2.78.51 2.9 1.71 E9-SE2 2.6 5.61 2.2 1.23 E9-SE3 2.5 6.75 2.1 1.25 E9-SE42.7 5.82 2.9 1.71 E9-SE5 2.6 6.38 2.4 1.76 E9-SE6 2.5 8.16 2.9 1.53E9-SE7 2.4 7.81 2.4 1.94 E9-SE8 2.5 9.25 2.6 2.3 E9.1 2.5 4.55 2.5 1.02

TABLE 14VH and VL amino acid sequences of human CDR-grafted E9 antibodies. SEQID No. Protein region Sequence 123456789012345678901234567890 334VH E9.1 EVQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCAREDVILRGGSDYWGQGTLVTVS S VH E9.1Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 334 VH E9.1 Residues 52-DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 334 VH P9.1 ResiduesEDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 334123456789012345678901234567890 335 VL E9.1SYELTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAW DRDTGVFGYGTRVTVLG VL E9.1 Residues 23-SGQRLGDKYAS CDR-L1 33 of SEQ ID NO.: 335 VL E9.1 Residues 49- EDSKRPSCDR-L2 55 of SEQ ID NO.: 335 VL E9.1 Residues 88- QAWDRDTGV CDR-L396 of SEQ ID NO.: 335 123456789012345678901234567890 336 VH E9-SE1EVQLVESGGGLVQPGGSLRLSCAVSGGSIS SSSYYWGWIRQAPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNSFYLQMNSLRAE DTAVYYCAREDVILRGGSDYWGQGTLVTVS CVH E9-SE1 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 336 VH E9-SE1Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 336 VH E9-SE1Residues 100- EDVILRGGSDY CDR-H3 110 of SEQ ID NO.: 336123456789012345678901234567890 337 VL E9-SE1LYVLTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQTMDEADYLCQAW DRDTGVFGGGTKVTVLG VL E9-SE1 Residues 23-SGQRLGDKYAS CDR-L1 33 of SEQ ID NO.: 337 VL E9-SE1 Residues 49- EDSKRPSCDR-L2 55 of SEQ ID NO.: 337 VL E9-SE1 Residues 88- QAWDRDTGV CDR-L396 of SEQ ID NO.: 337 123456789012345678901234567890 338 VH E9-SE2EVQLVESGGGLVQPGGSLRLSCAVSGGSIS SSSYYWGWIRQAPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNSFYLQMNSLRAE DTAVYYCAREDVILRGGSDYWGQGTLVTVS SVH E9-SE2 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 338 VH E9-SE2Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 338 VH E9-SE2Residues EDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 338123456789012345678901234567890 339 VL E9-SE2SYELTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAW DRDTGVFGYGTRVTVLG VL E9-SE2 Residues 23-SGQRLGDKYAS CDR-L1 33 of SEQ ID NO.: 339 VL E9-SE2 Residues 49- EDSKRPSCDR-L2 55 of SEQ ID NO.: 239 VL E9-SE2 Residues 88- QAWDRDTGV CDR-L396 of SEQ ID NO.: 339 123456789012345678901234567890 340 VH E9-SE3EVQLQESGPGLVKPGETLSLTCTVSGGSIS SSSYYWGWIRQAPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFYLKLSSVRAE DTAVYYCAREDVILRGGSDYWGQGTLVTVS SVH E9-SE3 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 340 VH E9-SE3Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 340 VH E9-SE3Residues EDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 340123456789012345678901234567890 341 VL E9-SE3SYELTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAW DRDTGVFGYGTRVTVLG VL E9-SE3 Residues 23-SGQRLGDKYAS CDR-L1 33 of SEQ ID NO.: 341 VL E9-SE3 Residues 49- EDSKRPSCDR-L2 55 of SEQ ID NO.: 341 VL E9-SE3 Residues 88- QAWDRDTGV CDR-L396 of SEQ ID NO.: 341 123456789012345678901234567890 342 VH E9-SE4EVQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCAREDVILRGGSDYWGQGTLVTVS SVH E9-SE4 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 342 VH E9-SE4Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 342 VH E9-SE4Residues EDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 342123456789012345678901234567890 343 VL E9-SE4LYVLTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQTMDEADYLCQAW DRDTGVFGGGTKVTVLG VL E9-SE4 Residues 23-SGQRLGDKYAS CDR-L1 33 of SEQ ID NO.: 343 VL E9-SE4 Residues 49- EDSKRPSCDR-L2 53 of SEQ ID NO.: 343 VL E9-SE4 Residues 88- QAWDRDTGV CDR-L396 of SEQ ID NO.: 343 123456789012345678901234567890 344 VH E9-SE5EVQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCAREDVILRGGSDYWGQGTLVTVS SVH E9-SE5 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 344 VH E9-SE5Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 344 VH E9-SE5Residues EDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 344123456789012345678901234567890 345 VL E9-SE5EYVLTQSPDFQSVTPKEKVTITCSGQRLGD KYASWYQQKPDQSPKLVIYEDSKRPSGVPSRFSGSNSGDDATLTINSLEAEDAATYYCQA WDRDTGVFGQGTKVEIKR VL E9-SE5 Residues 24-SGQRLGDKYAS CDR-L1 34 of SEQ ID NO.: 345 VL E9-SE5 Residues 50- EDSKRPSCDR-L2 56 of SEQ ID NO.: 345 VL E9-SE5 Residues 89- QAWDRDTGV CDR-L397 of SEQ ID NO.: 345 123456789012345678901234567890 346 VH E9-SE6EVQLQESGPGLVKPGETLSLTCTVSGGSIS SSSYYWGWIRQAPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFYLKLSSVRAE DTAVYYCAREDVILRGGSDYWGQGTLVTVS SVH E9-SE6 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 346 VH E9-SE6Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 346 VH E9-SE6Residues EDVILRGGSDY CDR-H3 100-110 SEQ ID NO.: 346123456789012345678901234567890 347 VL E9-SE6LYVLTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQTMDEADYLCQAW DRDTGVFGGGTKVTVLG VL E9-SE6 Residues 23-SGQRLGDKYAS CDR-L1 33 of SEQ ID NO.: 347 VL E9-SE6 Residues 49- EDSKRPSCDR-L2 55 of SEQ ID NO.: 347 VL E9-SE6 Residues 88- QAWDRDTGV CDR-L396 of SEQ ID NO.: 347 348 VH E9-SE7 EVQLVESGGGLVQPGGSLRLSCAVSGGSISSSSYYWGWIRQAPGKGLEWIGDIYYTGSTY YNPSLKSRVTISVDTSKNSFYLQMNSLRAEDTAVYYCAREDVILRGGSDYWGQGTLVTVS S VH E9-SE7 Residues 31- SSSYYWG CDR-H137 of SEQ ID NO.: 348 VH E9-SE7 Residues 52- DIYYTGSTYYNPSLKS CDR-H267 of SEQ ID NO.: 348 VH E9-SE7 Residues EDVILRGGSDY CDR-H3 100-110 ofSEQ ID NO.: 348 123456789012345678901234567890 349 VL E9-SE7EYVLTQSPDFQSVTPKEKVTITCSGQRLGD KYASWYQQKPDQSPKLVIYEDSKRPSGVPSRFSGSNSGDDATDTINSLEAEDAATYYCQA WDRDTGVFGQGTKVEIKR VL E9-SE7 Residues 24-SGQRLGDKYAS CDR-L1 34 of SEQ ID NO.: 249 VL E9-SE7 Residues 50- EDSKRPSCDR-L2 56 of SEQ ID NO.: 349 VL E9-SE7 Residues 89- QAWDRDTGV CDR-L397 of SEQ ID NO.: 349 123456789012345678901234567890 350 VH E9-SE8EVQLQESGPGLVKPGETLSLTCTVSGGSIS SSSYYWGWIRQAPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFYDKLSSVRAE DTAVYYCAREDVILRGGSDYWGQGTLVTVS SVH E9-SE8 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 350 VH E9-SE8Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 350 VH E9-SE8Residues EDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 350123456789012345676901234567890 351 VL E9-SE8EYVLTQSPDFQSVTPKEKVTITCSGQRLGD KYASWYQQKPDQSPKLVIYEDSKRPSGVPSRFSGSNSGDDATLTINSLEAEDAATYYCQA WDRDTGVFGQGTKVEIKR VL E9-SE8 Residues 24-SGQRLGDKYAS CDR-L1 34 of SEQ ID NO.: 351 VL E9-SE8 Residues 50- EDSKRPSCDR-L2 56 of SEQ ID NO.: 351 VL E9-SE8 Residues 89- QAWDRDTGV CDR-L397 of SEQ ID NO.: 351

TABLE 15 Summary of the E9 antibody variants generated and expressiondata. HEK-293-6e titer Antibody VH name VL name (mg/L) E9-FR1 E9VH325E9VL325 29 E9-FR2 E9VH1D4.1 E9VL1D4.1 44

TABLE 16 Binding affinities of the E9 CDR-grafted antibodies for human,mouse and cynomolgus DLL4 as determined by ELISA, Biacore and FACS.Binding Data Human DLL4 Mouse DLL4 Cyno DLL4 Binding Binding BindingELISA Biacore Binding ELISA Biacore Binding ELISA Biacore (EC50, (Kd,FACS (EC50, (Kd, FACS (EC50, (Kd, MAb nM) nM) (Kd, nM) nM) nM) (Kd, nM)nM) nM) E9-FR1 0.11 0.72 4 0.13 1.6 2.51 0.12 0.74 E9-FR2 0.105 0.31 6.20.125 0.69 3.57 0.12 0.3 E9.1 0.03 0.52 3.62 0.04 1.1 1.66 0.035 0.47

TABLE 17 Neutralizing activities of the E9 CDR-grafted antibodies forhuman and mouse DLL4 as determined by ELISA and FACS. Functional DataHuman DLL4 Mouse DLL4 Blocking Blocking Blocking Blocking ELISA FACSELISA FACS MAb (IC50, nM) (IC50, nM) (IC50, nM) (IC50, nM) E9-FR1 1.541.57 3.35 0.98 E9-FR2 1.52 3.03 4.35 1.07 E9.1 2.2 1.62 5.85 1.06

TABLE 18VH and VL amino acd sequences of human CDR-grafted E9 antibodies. SEQ IDNo. Protein region Sequence 123456789012345678901234567890 352 VH E9-FR1EVQLVQSGTEVKKPGESLKISCKVSGGSIS SSSYYWGWIRQMPGKGLEWIGDIYYTGSTYYNPSLKSQVTISVDTSFNTFFLQWSSLKAS DTAMYYCAREDVILRGGSDYWGQGTMVTVS SVH E9-FR1 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 352 VH E9-FR1Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 352 VH E9-FR1Residues EDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 352123456789012345678901234567890 353 VL E9-FR1EYVLTQSPATLSVSPGERATLSCSGQRLGD KYASWYQQKPGQSPRLVIYEDSKRPSDIPARFSGSNSGDEATLTISSLQSEDFAVYYCQA WDRDTGVFGQGTRLEIKR VL E9-FR1 Residues 24-SGQRLGDKYAS CDR-L1 34 of SEQ ID NO.: 353 VL E9-FR1 Residues 50- EDSKRPSCDR-L2 56 of SEQ ID NO.: 353 VL E9-FR1 Residues 89- QAWDRDTGV CDR-L397 of SEQ ID NO.: 35 123456789012345676901234567890 354 VH E9-FR2EVTLRESGPALVKPTQTLTLTCTVSGGSIS SSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFVLTMTNMDPV DTATYYCAREDVILRGGSDYWGQGTTVTVS SVH E9-FR2 Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 354 VH E9-FR2Residues 52- DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 354 VH E9-FR2Residues EDVILRGGSDY CDR-H3 100-110 of SEQ ID NO.: 354123456789012345678901234567890 355 VL E9-FR2DYVLTQSPDSLAVSLGERATINCSGQRLGD KYASWYQQKPGQSPKLVIYEDSKRPSGIPDRFSGSNSGDDATLTISSLQAEDVAVYYCQA WDRDTGVFGGGTKVEIKR VL E9-FR2 Residues 24-SGQRLGDKYAS CDR-L1 34 of SEQ ID NO.: 355 VL E9-FR2 Residues 50- EDSKRPSCDR-L2 56 of SEQ ID NO.: 355 VL E9-FR2 Residues 89- QAWDRDTGV CDR-L397 of SEQ ID NO.: 355

Example 6: Further Engineering of Affinity-Matured Antibody E9-71E9-71(M) and E9-71(L).

Both heavy chain and light chain of affinity-matured anti-DLL4 antibodyE9-71 were further engineered. The methionine (M) in CDR-H3 of E9-71heavy chain was mutated to leucine (L) by designing forward and reverseoverlapping primers containing the mutated nucleotides. Polymerase chainreaction (PCR) was performed in two sequential steps to amplify theentire variable region gene using the two primers carrying the mutatednucleotides and two outermost primers containing overhanging sequencescomplementary to the receiving vector.

The signal peptide used for the light chain of E9-71(M) and E9-71(L) iscalled lambda 1a signal peptide. The framework 4 (FW4) region hJL-1 ofE9-71 light chain was changed to hJL2 to be more compatible with thehuCL2 constant region of the antibody. Forward and reverse primers weredesigned containing the mutated nucleotides. Polymerase chain reaction(PCR) was performed in two sequential steps to amplify the entirevariable region gene using the two primers carrying the mutatednucleotides and two outermost primers containing overhanging sequencescomplementary to the receiving vector.

The PCR products derived from each cDNA assembly was separated on anagarose gel and the band corresponding to the predicted variable regioncDNA size excised and purified. The variable heavy region were insertedin-frame onto a cDNA fragment encoding the human IgG1 constant regioncontaining two hinge-region amino acid mutations by homologousrecombination in bacteria. These mutations are a leucine to alaninechange at position 234 (EU numbering) and a leucine to alanine change atposition 235 (Lund et al., J. Immunol., 147: 2657 (1991)). The variablelight chain region were inserted in-frame between the human lambda 1asignal peptide and the human lambda constant region by homologousrecombination. Bacterial colonies were isolated and plasmid DNAextracted; cDNA inserts were sequenced in their entirety. Correct heavyand light chains corresponding to each antibody (Table 19) wereco-transfected into HEK-293-6E cells to transiently produce full-lengthE9-71(M) or E9-71(L) anti-human DLL4 antibodies. Both E9.71(M) andE9.71(L) share the same light chain. Cell supernatants containingrecombinant human antibody may be purified by Protein A Sepharosechromatography and bound antibody eluted by addition of acid buffer.Antibodies were neutralized and dialyzed into PBS.

TABLE 19VH and VL Amino Acid Sequences of Human E9.71 engineered antibodies SEQID No. Protein region Sequence 123456789012345678901234567890 356VH E9.71 EVQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCARQALAMGGGSDKWGQGTLVTVS S VH E9.71Residues 31- SSSYYWG CDR-H1 37 of SEQ ID NO.: 356 VH E9.71 Residues 52-DIYYTGSTYYNPSLKS CDR-H2 67 of SEQ ID NO.: 356 VH E9.71 ResiduesQALAMGGGSDK CDR-H3 100-110 of SEQ ID NO.: 356123456789012345678901234567890 357 VL E9.71SYELTQPPSVSVSPGQTASITCSGQRLGDK YASWYYQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAW DRDTGVFGYGTKVTVLG VL E9.71 Residues 23-SGQRLGDKYAS CDR-L1 33 of SEQ ID NO.: 357 VL E9.71 Residues 49- EDSKRPSCDR-L2 55 of SEQ ID NO.: 357 VL E9.71 Residues 88- QAWDRDTGV CDR-L396 of SEQ ID NO.: 357 123456789012345678901234567890 358 VH E9.71(M)EVQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWGWIRQPPGKGLEWIGDIYYTGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAA DTAVYYCARQALAMGGGSDKWGQGTLVTVS S VHResidues 31- SSSYYWG E9.71(M) 37 of SEQ ID CDR-H1 NO.: 358 VHResidues 52- DIYYTGSTYYNPSLKS E9.71(M) 67 of SEQ ID CDR-H2 NO.: 358 VHResidues QALAMGGGSDK E9.71(M) 100-110 of CDR-H3 SEQ ID NO.: 358123456789012345678901234567890 359 VL E9.71(M)SYELTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAW DRDTGVFGGGTKLTVLG VL Residues 23-SGQRLGDKYAS E9.71(M) 33 of SEQ ID CDR-L1 NO.: 359 VL Residues 49-EDSKRPS E9.71(M) 55 of SEQ ID CDR-L2 NO.: 359 VL Residues 88- QAWDRDTGVE9.71(M) 96 of SEQ ID CDR-L3 NO.: 359 123456789012345678901234567890 360VH E9.71(L) EVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGDIYYTGSTY YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQALALGGGSDKWGQGTLVTVS S VH Residues 31- SSSYYWG E9.71(L)37 of SEQ ID CDR-H1 NO.: 360 VH Residues 52- DIYYTGSTYYNPSLKS E9.71(L)67 of SEQ ID CDR-H2 NO.: 360 VH Residues QALALGGGSDK E9.71(L) 100-110 ofCDR-H3 SEQ ID NO.: 360 123456789012345678901234567890 361 VL E9.71(L)SYELTQPPSVSVSPGQTASITCSGQRLGDK YASWYQQKPGQSPVLVIYEDSKRPSGIPERFSGSNSGDTATLTISGTQPMDEADYYCQAW DRDTGVFGGGTKLTVLG VL Residues 23-SGQRLGDKYAS E9.71(L) 33 of SEQ ID CDR-L1 NO.: 361 VL Residues 49-EDSKRPS E9.71(L) 55 of SEQ ID CDR-L2 NO.: 361 VL Residues 88- QAWDRDTGVE9.71(L) 96 of SEQ ID CDR-L3 NO.: 361

Example 7: E9-71(M) Signal Peptide Engineering

Signal peptide lambda 1a was used for the generation of anti-DLL4antibody E9-71(M). Alternative signal peptides were also investigated.The prediction of the percentage of the correct antibody cleavage duringmammalian expression of in silico constructed amino acid sequences ofE9-71(M) N-terminal variable region with different signal peptides fromthe lambda and kappa signal peptide families is known in the art usingthe Signal IP 3.0 Server available on the Internet (e.g., worldwidewebsite cbs.dtu.dk/services/SinalP/) or numerous other equivalentsoftware. The signal peptides with the highest predicted percentages ofcorrect cleavage, one from each family were chosen: the lambda 3p fromthe lambda family and the L23 from the kappa family. A mutated versionof the original lambda 1a signal peptide was also selected, with twoamino acid changes (glycine to arginine and serine to valine). For theconstruction of the light chain containing the lambda 1a signal peptidepolymerase chain reaction (PCR) was performed in one step to amplify theentire variable region gene using the two outermost primers containingoverhanging sequences complementary to the receiving vector with one ofthem containing the mutated nucleotide sequence. For the construction ofthe light chain containing the lambda 3p signal peptide and the kappaL23 signal peptide two overlapping primers were designed to constructthe signal peptide region then polymerase chain reaction (PCR) wasperformed to amplify the entire variable region gene using the twooutermost primers containing overhanging sequences complementary to thereceiving vector. Two versions of the E9-71 variable regions weregenerated using the lambda 3p and the kappa L23 signal peptides: onewith the full length variable region and another one with the firstserine (S) at the variable region N-terminus missing. Only the fulllength variable region was generated with the lambda 1a signal peptide.The PCR products derived from each cDNA assembly were separated on anagarose gel and the band corresponding to the predicted variable regioncDNA size excised and purified. The variable heavy region was insertedin-frame onto a cDNA fragment encoding the human IgG1 constant regioncontaining two hinge-region amino acid mutations by homologousrecombination in bacteria. These mutations are a leucine to alaninechange at position 234 (EU numbering) and a leucine to alanine change atposition 235 (Lund et al., J. Immunol., 147: 2657 (1991)). The variablelight chain region was inserted in-frame with the human lambda constantregion by homologous recombination. Bacterial colonies were isolated,plasmid DNA extracted, and cDNA inserts were sequenced in theirentirety. Correct heavy and light chains corresponding to each antibodywere co-transfected into HEK-293-6E cells to transiently producefull-length E9-71(M) anti-human DLL4 antibodies. Cell supernatantscontaining recombinant human antibody were purified by Protein ASepharose chromatography and bound antibody eluted by addition of acidbuffer. Antibodies were dialyzed into PBS. Purified E9.71(M) antibodieswere analyzed by Mass Spectrometry (MS) for confirmation of intactantibody sequence. Table 20, below, shows the amino acid sequence of thedifferent signal peptides used for the generation of E9.71(M). Table 21,below, shows the E9.71(M) cleavage sites analyzed by Mass Spectrometry.

TABLE 20Amino acid sequence of the signal peptides used for the generationof E9.71(M) Antibody Signal Peptide Signal Peptide Sequence VL RegionE9.71(M) λ 1a MAWSPLFLTLITHCAGSWA Full Length (SEQ ID NO: 362)E9.71(M)-1 λ 1a MAWSPLFLTLITHCARVWA Full Length (GS to RV)(SEQ ID NO: 363) E9.71(M)-2 λ 3p MAWTPLLLPLLTFCTVSEA Full Length(SEQ ID NO: 364) E9.71(M)-3 λ 3p MAWTPLLLPLLTFCTVSEA N-terminus S(SEQ ID NO: 365) missing E9.71(M)-4 Kappa L23 MDMRVPAQRLGLLLLWFPGARCFull Length (SEQ ID NO: 366) E9.71(M)-5 Kappa L23 MDMRVPAQRLGLLLLWFPGARCN-terminus S (SEQ ID NO: 367) missing

TABLE 21E9.71(M) antibody cleavage sites analyzed by Mass Spectrometry (MS)% Major Antibody Antibody Major Cleavage Site Peak (↓) E9.71(M)MAWSPLFLTLITHCAG↓SWA↓SYELTQPPSVS 95 (SEQ ID NO: 368) E9.71(M)-1MAWSPLFLTLITHCARVWA↓SYELTQPPSVS 96 (SEQ ID NO: 369) E9.71(M)-2MAWTPLLLPLLTFCTVSEA↓SYELTQPPSVS 96 (SEQ ID NO: 370) E9.71(M)-3MAWTPLLLPLLTFCTVSEA↓YELTQPPSVS 97 (SEQ ID NO: 371) E9.71(M)-4MDMRVPAQRLGLLLLWFPGARC↓SYELTQPPSVS Not (SEQ ID NO: 372) determinedE9.71(M)-5 MDMRVPAQRLGLLLLWFPGARC↓YELTQPPSVS Not (SEQ ID NO: 373)determined

Example 8. In Vitro Characterization of Engineered PROfusion Antibodies

The antigen binding affinities of these engineered PROfusion antibodieswere determined by the BIACORE technology as described in Example 1.1,and are shown in Table 22. The in vitro activities of the representativeones were further evaluated using other methods described in Example 1,with results shown in Table 23.

TABLE 22 Biacore kinetics of engineered anti-DLL4 PROfusion antibodies.Binding kinetics Human DLL4 ECD Mouse DLL4 ECD Cyno DLL4 ECD K_(a) K_(d)K_(a) K_(d) K_(a) K_(d) (M⁻¹S⁻¹) (S⁻¹) K_(D) (M⁻¹S⁻¹) (S⁻¹) K_(D)(M⁻¹S⁻¹) (S⁻¹) K_(D) Antibody E+04 E−05 (nM) E+04 E−05 (nM) E+04 E−05(nM) E9.4 5.67 3.31 0.58 5.38 6.01 1.12 1.37 3.44 0.25 E9.11 3.63 1.740.48 2.77 1.39 0.5 5.31 0.89 0.17 E9.14 3.99 36.5 9.14 3.89 13.6 3.495.72 38.6 6.75 E9.17 4.39 0.44 0.1 3.7 1.41 0.38 7.51 1.23 0.16 E9.181.57 1.84 1.18 2.32 1.68 0.73 2.63 2.63 1 E9.19 7.49 5.17 0.69 6.7 20.63.07 1.92 4.86 0.25 E9.22 3.91 33.1 8.46 3.78 13.3 3.5 5.62 35.2 6.26E9.48 1.1 7.59 6.92 1.65 4.17 2.53 1.68 7.91 4.71 E9.65 2.59 36.3 142.57 13.6 5.27 3.21 41.4 12.9 E9.66 3.26 0.25 0.078 2.89 1.44 0.50 6.550.91 0.14 E9.71 3.88 3.51 0.91 3.55 21.6 6.08 7.75 3.79 0.49 E9.13 3.744.97 1.33 4.34 21.2 4.89 7.92 4.95 0.63 E9.16 1.29 3.15 2.45 1.83 14.88.1 2.49 3.79 1.52 E9-38 1.27 3.48 2.75 1.84 17.1 9.27 2.42 3.47 1.44E9.2B 35.6 0.79 0.02 N/D N/D N/D N/D N/D N/D E9.IF 21.8 1.16 0.05 N/DN/D N/D N/D N/D N/D E9-12B 15.1 0.31 0.02 8.35 1.47 0.18 N/D N/D N/DE9-10H 10.4 5.9 0.57 6.53 2.23 0.34 N/D N/D N/D E9-5E 15 0.6 0.04 8.90.2 0.02 N/D N/D N/D E9-10C 12 4.59 0.38 7.37 12.1 1.6 N/D N/D N/DE9-10E 21.8 4.62 0.21 15.1 9.41 0.62 N/D N/D N/D E9-7E 15.2 4.12 0.279.53 22.1 2.3 N/D N/D N/D A10.K30 3.57 27.4 7.69 NB NB NB N/D N/D N/DA10.K42 10 40.6 4.06 NB NB NB N/D N/D N/D A10.L45 4.4 3.16 0.72 NB NB NBN/D N/D N/D A10.L73 5.07 0.95 0.19 NB NB NB N/D N/D N/D MAb = monoclonalantibody; N/D = not determined, NB = no binding

TABLE 23 Characterization of selected engimered PROfusion DLL4 antibody.Functional Blockade Assays Competition Competition Inhibition of DirectBinding Assays ELISA FACS Notch Capture ELISA FACS (IC₅₀, nM) (IC₅₀, nM)activation via (EC₅₀, nM) (EC₅₀, nM) DLL4 ECD/ huNotch-1/ huDLL4 cells,DLL4 ECD DLL4 Cells huNotch-1 DLL4 cells coculture Antibody hu mu cynohu mu hu mu cyno hu mu (IC₅₀, nM) E9-2B 0.16 0.16 0.45 1.78 0.35 2.3 2.82.2 2.63 0.34 2.1 E9-71 0.16 0.17 0.44 1.82 0.32 2.0 1.3 2.0 3.22 0.242.1 E9-19 0.17 0.17 0.46 2.79 0.54 1.8 1.3 1.5 4.90 0.44 4.4 E9-4 N/DN/D N/D 1.63 0.63 N/D N/D N/D 4.3 0.3 N/D E9-11 N/D N/D N/D 4.46 1.45N/D N/D N/D 14.75 2.06 N/D E9-16 N/D N/D N/D 1.7 >50 N/D N/D N/D 12.61.1 N/D E9-17 N/D N/D N/D 8 2.57 N/D N/D N/D 19.79 N/D 1 E9-22 N/D N/DN/D 34.34 37.35 N/D N/D N/D 21.05 2.63 N/D E9-38 N/D N/D N/D 1.66 0.13N/D N/D N/D 7.76 1.2 N/D E9-48 N/D N/D N/D N/D N/D N/D N/D N/D 9.12 1.3N/D E9-66 N/D N/D N/D 2.02 0.7 N/D N/D N/D 5.03 0.99 N/D E9-1F N/D N/DN/D 2.65 0.37 N/D N/D N/D 6.55 0.65 N/D E9-5E N/D N/D N/D 2.7 0.89 N/DN/D N/D 3.76 0.34 N/D E9-7E N/D N/D N/D 4.46 1.94 N/D N/D N/D 4 0.330.41 E9-10C N/D N/D N/D 3.15 0.88 N/D N/D N/D 3.03 0.26 0.85 E9-10E N/DN/D N/D 5.1 1.19 N/D N/D N/D 3.83 0.36 N/D E9-10H N/D N/D N/D 3.52 0.82N/D N/D N/D 5.65 0.6 N/D E9-12B N/D N/D N/D 3.34 0.71 N/D N/D N/D 4.440.37 N/D E9-71(M) N/D N/D N/D 0.27 0.29 N/D N/D N/D 2.12 0.18 N/DE9-71(L) N/D N/D N/D 0.22 0.47 N/D N/D N/D 1.82 0.21 2.4 E9-71(M)-3 N/DN/D N/D N/D N/D N/D N/D N/D N/D N/D 0.65 hu = human; mu = murine; cyno =cynomolgus monkey; N/D = not determined

Example 8: Physicochemical Properties of Selected PROfusion Antibodies.

The identity of monoclonal antibodies specific to DLL4 was determined bymass spectrometry as below.

Mass Spectrometry Analysis of E9-71.

Light chain and heavy chain molecular weight analysis: E9-71 sample wasdiluted to 1 mg/mL with Milli-Q water, 1 μL of 1 M DTT was added to 20μL of diluted sample. The sample was incubated at 37° C. for 30 minutes,1 μL of the reduced samples was injected onto the Agilent 6510 Q-TOFLC/MS system with a Varian Diphenyl column. Buffer A was 0.02%trifluoroacetic acid (TFA), 0.08% formic acid (FA) in water. Buffer Bwas 0.02% TFA, 0.08% FA in acetonitrile. The gradient started at 5% B,increased to 35% B in 5 minutes, and increased to 38% B in 15 minutes.The gradient then increased to 95% B in 1 minute and stayed at 95% B for4 minutes, and decreased to 5% B in 1 minute. The flow rate was 50μL/min. The mass spectrometer was operated at 5 kvolts spray voltage andscan range was from 600 to 3200 mass to charge ratio. The light chainmolecular weight of 22649 Dalton matched well with the theoretical valuewith the amino acid Y being the N-terminus. Three minor peaks wereobserved at molecular weight 22014 Dalton, 22737 Dalton, and 22937Dalton. The peak at 22014 Dalton was consistent with N-terminal −6 aminoacid fragment. This peak was most probably caused by the in-sourcefragmentation from the full length light chain, as lowering the massspectrometer “fragmentor value” can lead to the disappearance of thispeak. The 22737 Dalton was consistent with the theoretical value withthe amino acid S being the N-terminus. The 22937 Dalton was consistentwith the light chain with signal peptide extension of amino acids “LS”on the N-terminal. The heavy chain molecular weights matched well withthe theoretical values. The observed molecular weights were 50263Dalton, 50426 Dalton, and 50588 Dalton, with the differencecorresponding to 162 Dalton as result of different glycosylation.

Mass Spectrometry Analysis of E9-71(M).

The same method described in “Mass spectrometry analysis of E9-71” wasused to analyze the E9-71(M) sample. The light chain molecular weight of22645 Dalton matched well with the theoretical value with the amino acidS being the N-terminus. A small peak with molecular weight 22989 Daltonwas observed, corresponding to the light chain with signal peptideextension of amino acids “SWA” on the N-terminal. A very small peak withmolecular weight 21923 Dalton was also observed, although it was highlylikely caused by in-source fragmentation as lowering the massspectrometer “fragmentor value” led to disappearance of this peak. Theheavy chain molecular weights matched well with the theoretical values.The observed molecular weights were 50263 Dalton, 50426 Dalton, and50588 Dalton, with the difference corresponding to 162 Dalton as resultof different glycosylation.

Mass Spectrometry Analysis of E9-71 (L).

The same method described in “Mass spectrometry analysis of E9-71” wasused to analyze the E9-71(L) sample. The light chain molecular weight of22645 Dalton matched well with the theoretical value with the amino acidS being the N-terminus. A small peak with molecular weight 22989 Daltonwas observed, corresponding to the light chain with signal peptideextension of amino acids “SWA” on the N-terminal. A very small peak withmolecular weight 21923 Dalton was also observed, although it was highlylikely caused by in-source fragmentation as lowering the massspectrometer “fragmentor value” led to disappearance of this peak. Theheavy chain molecular weights matched well with the theoretical values.The observed molecular weights were 50245 Dalton, 50407 Dalton, and50569 Dalton, with the difference corresponding to 162 Dalton as resultof different glycosylation.

Mass Spectrometry Analysis of E9-71(M)-3.

The same method described in “Mass spectrometry analysis of E9-71” wasused to analyze the E9-71(M)-3 sample. The light chain molecular weightof 22558 Dalton matched well with the theoretical value. A very smallpeak with molecular weight 21923 Dalton was also observed, although itwas highly likely caused by in-source fragmentation as lowering the massspectrometer “fragmentor value” led to disappearance of this peak. Theheavy chain molecular weights matched well with the theoretical values.The observed molecular weights were 50263 Dalton, 50426 Dalton, and50588 Dalton, with the difference corresponding to 162 Dalton as resultof different glycosylation.

The solubilities of the antibodies were estimated by polyethylene glycol(PEG) 3000 precipitation. They were also directly determined, i.e., realsolubility, by concentrating the antibodies in a specific solutionand/or buffer with Amicon centrifugal filters and then observed for anyprecipitation at 25° C., and 5° C. Stability was inferred by nearultra-violet circular (UV-CD) and differential scanning calorimetry(DSC). Stability to freezing and thawing and at elevated temperatures(accelerated stability) was assessed by size exclusion chromatography(SEC). The detail techniques were described in Example 1.8 and theresults are described below: Solubility Estimation by PEG PrecipitationResults.

Tables 24 and 26 shows the percentage of PEG 3000 needed to induceprecipitation for a series of E9 clones. The clones and the Adalimumnabreference were formulated at 0.2 mg/ml. According to the results, clonessuch as E9-4. E9-14. E9-22, and E9-19 are estimated to have solubilitiessimilar to that of adalimumab (approximately 200 mg/ml) while clonessuch as E9-11 and E9-17 are estimated to have much lower solubilities.

Table 25 shows the percentage of PEG 3000 needed to induce precipitationfor a series of stability engineered E9 clones. The clones and theadalimumab reference were formulated at 0.2 mg/ml. According to theresults, clones such as E9-SE1 have the highest solubilities in theseries but are not expected to have solubilities similar to that ofadalimumab (approximately 200 mg/ml) while clones such as E9-SE5 areestimated to have much lower solubilities.

TABLE 24 Percentage of PEG 3000 needed to induce precipitation for aseries of E9 antibodies. (The antibodies were formulated at 0.2 mg/ml.)A-Number Lot # Antibody % PEG 3000 A-1242367.0 1718299 DLL4-E9-11hIgG1/L 3.00 A-1242368.0 1718300 DLL4-E9-17 hIgG1/L 3.00 E9.1 IgG2 3.00A-1242369.0 1718301 DLL4-E9-18 hIgG1/L 4.00 A-1242370.0 1718302DLL4-E9-48 hIgG1/L 4.00 E9.1 IgG4 5.00 DLL4-E9-3 hIgG/L 6.00 A-1242371.01718303 DLL4-E9-66 hIgG1/L 8.00 A-1241120.0 1716682 DLL4-E9-16 hIgG1/L10.00 A-1242795.0 1718785 DLL4-E9-13 hIgG1/L 10.00 A-1241121.0 1716683DLL4-E9-38 hIgG1/L 11.00 A-1242800.0 1718790 DLL4-E9-71 hIgG1/L 11.00A-1242794.0 1718784 DLL4-E9-4 hIgG1/L 12.00 A-1242796.0 1718786DLL4-E9-14 hIgG1/L 12.00 A-1242798.0 1718788 DLL4-E9-22 hIgG1/L 12.00A-1242797.0 1718787 DLL4-E9-19 hIgG1/L 14.00 adalimumab 14.00

TABLE 25 Percentage of PEG 3000 needed to induce precipitation for aseries of E9 stability engineered antibodies. (The antibodies wereformulated at 0.2 mg/ml.) Antibody % PEG 3000 E9-SE5 6.00 E9-SE7 7.00E9-SE4 7.50 E9-SE8 7.50 E9-SE2 9.00 E9-SE3 9.00 E9-SE6 9.50 E9-SE1 10.00(+) E9.1 9.00 (+) E9   10.50 adalimumab 13.00

TABLE 26 Percentage of PEG 3000 needed to induce precipitation for aseries of E9 antibodies. (The antibodies were formulated at 0.2 mg/ml.)Antibody % PEG 3000 E9-2B 8.00 E9-1F 8.00 E9 10 adalimumab 14

Real Solubility Screening Results: E9. E9-19. E9-71, E9-2B, E9-1F.

Solutions containing 12 mg of E9, E9-71. E9-1F, and E9-19 and 5.5 mg ofE9-2B were obtained. E9 is the IgG1 mutant isotype. The volumes of allsolutions were reduced below 1 ml by ultra-centrifugation with Amicon30K 15 ml tubes.

After this step, the following was observed: E9-2B and E9-19 were clear.E9. E9-71, and E9-1F were slightly cloudy.

10 ml of 15 mM histidine buffer at pH 5.03 was added to each tube andthe solutions re-concentrated to 1 ml. The solutions were thentransferred to Amicon 30K 4 ml tubes and concentrated to as low a volumeas possible.

After this step, the following was observed at room temperature:

-   -   E9: 163 mg/ml; vol=0.05 ml; pH=5.12    -   E9-19: 132 mg/ml; vol=0.05 ml: pH=5.06    -   E9-71: 193 mg/ml; vol=0.05 ml; pH=5.32    -   E9-2B: 64 mg/ml; vol=0.1 ml: pH=5.29    -   E9-1F: 100 mg/ml; vol=0.1 ml; pH=5.31

Both E9-2B and E9-1F required much longer to concentrate than the otherthree. This may suggest that their viscosity is high under theformulation conditions.

The solutions were then placed at 5° C. for two days to assesssolubility at this temperature. The following was observed:

-   -   E9: remained clear at 5° C., and when brought back to room        temperature    -   E9-71: remained clear at 5° C. and when brought back to room        temperature    -   E9-1F: showed an extremely slight amount of cloudiness at 5° C.        that cleared when brought to room temperature after 20 minutes    -   E9-2B: showed a slight amount of cloudiness at 5° C. that        cleared when brought to room temperature after 20 minutes    -   E9-19: showed apparent cloudiness at 5° C. that cleared when        brought to room temperature after 20 minutes.

Real Solubility Screening Results for E9-71.

For E9-71, 4 mg in solution was concentrated with Amicon centrifugalfilters to 60 mg/ml. No precipitation or cloudiness was observed at 25°C. nor after storage for 1 day at 5° C.

Tertiary Structure Characterization by Near UV-CD Results.

Near UV-CD was performed on E9, E9-19, E9-71, E9-2B, and E9-1F samplesat 1 mg/ml. The profiles of the spectra show a sigmoidal patterntypically observed for properly folded antibodies. From this technique,no indication of misfolding is observed for the antibodies. IntrinsicStability Characterization by Differential Scanning Calorimetry (DSC).

DSC was performed on E9. E9-19. E9-71. E9-2B, and E9-1F samples at 1mg/ml. The results are given in Table 27. The onset is the temperatureat which unfolding initiates. Also, an IgG antibody typically showsthree unfolding transitions (Tm): unfolding of the intact antibody isassociated with the melting of the CH2 domain in the Fc fragment,melting of the CH3 domain in the Fc fragment, and melting of the Fabfragment. Onset values suggest E9, E9-19, and E9-71 are most stable.Typically, clones with higher Tm values are preferred over those withlower values.

TABLE 27 Intrinsic stability of anti-DLL4 E9 clones via DSC at 1 mg/ml.Antibody Tm1 (° C.) Tm2 (° C.) Tm3 (° C.) Onset (° C.) E9 64.45 72.1280.04 54 E9-19 65.66 77 — 56 E9-71 65.65 75.36 81.24 54 E9-2B 64.7480.58 83.27 52.5 E9-1F 63.84 80.51 83.26 51.3

Evaluation of Stability to Freeze-Thaw Stress

Stability to freeze-thaw stress was evaluated for E9, E9-19, E9-71,E9-2B, and E9-1F samples at 1 mg/ml. Table 28 shows the results of SECanalysis of samples after five freeze-thaw cycles. All the antibodiestested were stable to freeze-thaw stress. No apparent loss of monomerwas observed even after 5 freeze-thaw cycles.

TABLE 28 Percentage of monomer species quantitated by SEC beforefreezing at −80° C. and after five cycles of freezing at −80° C. andthawing at 30° C. in water bath for anti-DLL4 clones. % monomer after5th Antibody % monomer before freezing freeze-thaw cycle E9 99.2 99.3E9-19 99.3 99.1 E9-71 96.9 96.8 E9-2B 99.0 98.9 E9-1F 98.6 98.5

Evaluation of Stability at Elevated Temperatures (AcceleratedStability).

Stability at elevated temperatures was evaluated for E9. E9-19, E9-71,E9-2B, and E9-1F samples at 1 mg/ml. Table 29 shows the results of SECanalysis of samples at time zero and after 7 and 21 days at 40° C., and50° C. The degradation kinetics for all of the antibodies revealed anapproximately 8% reduction in monomer percentage after 21 days 50° C.

TABLE 29 Percentage of different species quantitated by SEC for anti-DLL4 clones at time zero and after incubation for 7 and 21 days at 40°C. and 50° C. (Samples were formulated at 1 mg/ml.) % % % % % monomermonomer monomer monomer monomer Anti- at time at 7 days at 7 days at 21days at 21 days body zero at 40° C. at 50° C. at 40° C. at 50° C. E999.2 98.6 86.8 90.9 88.1 E9-19 99.3 98.7 96.8 97.4 91.9 E9-71 96.9 96.495.4 95.0 91.0 E9-2B 99.0 98.6 97.0 97.1 92.4 E9-1F 98.6 98.2 96.5 96.891.9 % % % % % aggregate aggregate aggregate aggregate aggregate Anti-at time at 7 days at 7 days at 21 days at 21 days body zero at 40° C. at50° C. at 40° C. at 50° C. E9 0.3 0.3 0.3 0.5 0.6 E9-19 0.3 0.3 0.4 0.71.4 E9-71 2.4 2.3 1.8 2.9 7.8 E9-2B 0.5 0.4 0.5 0.9 1.7 E9-1F 0.3 0.30.3 0.6 1.6 % % % % % fragment fragment fragment fragment fragment Anti-at time at 7 days at 7 days at 21 days at 21 days body zero at 40° C. at50° C. at 40° C. at 50° C. E9 0.5 1.2 12.9 8.7 11.3 E9-19 0.4 1.0 2.81.9 6.7 E9-71 0.7 1.3 2.8 2.1 6.2 E9-2B 0.6 1.0 2.5 2.0 5.9 E9-1F 1.11.5 3.2 2.5 6.5

Example 9: Rodent PK Assessment of Anti-DLL4 Antibodies

To assess pharmacokinetics properties of anti-DLL4 antibodies,SCID-Beige mice (n=3 per antibody) were administered a singleintraperitoneal (IP) dose of antibody at either 5 or 30 mg/kgconcentration, depending on cross-reactivity of antibody to murine DLL4.Longitudinal serum samples (5 μl of whole blood diluted 1:50 inHBS-EP+buffer per time point) were collected from each animal over 21days. Serum concentrations were determined using a DLL4-specific Biacoreplatform. Briefly, human DLL4 was immobilized to a sensorchip andsamples were injected over the flowcell at 5 μl per minute for 5 minuteswith the resulting binding levels measured and compared to standards.Serum concentration time profiles were used to estimate thepharmacokinetic parameters of C_(max) (peak serum concentration), CL(clearance), and t_(1/2) (antibody half life), summarized in Table 30.For both E9 and A10 PROfusion antibodies, their pharmacokineticsproperties were improved through CDR-engineering during the process ofaffinity maturation.

TABLE 30 Pharmacokinetic parameters of anti-DLL4 antibodies inSCID-beige mice. Dose Cmax CL t_(1/2) Antibody (mg/kg) (μg/mL)(mL/hr/kg) (d) E9 30 201 3.10 1.3 E9-10C 30 165 0.82 5.2 E9-10E 30 2630.97 3.8 E9-10H 30 235 1.49 3.1 E9-12B 30 146 1.32 3.8 E9-19 30 179 1.084.4 E9-1F 30 269 0.89 4.2 E9-2B 30 165 0.65 5.5 E9-5E 30 234 0.95 3.0E9-66 30 114 2.90 1.9 E9-71 30 102 1.10 4.2 E9-71(L) 30 145 0.83 4.1E9-71(M) 30 113 1.07 4.3 E9-7E 30 130 1.23 5.8 A10 5 10.5 5.80 3.1A10.K30 5 15.6 0.69 17.7 A10.K42 5 12.5 0.93 13.8 A10.L45 5 18.6 0.6513.4

Example 10: Anti-DLL4 Antibody Treatment Increased Endothelial CellSprouting In Vitro

Fibrin gel beads sprouting assay was carried out to examine the in vitroangiogenesis activity of HUVEC (passage 2-3, Lonza) as described(Nakatsu et al., Microvasc. Res., 66: 102-112 (2003)). Briefly,fibrinogen solution was reconstituted with aprotinin (4 U/ml) andthrombin (50 U/ml). Cytodex 3 beads (Amersham Pharmacia Biotech) werecoated with 350 to 400 HUVECs per bead for overnight. About 20HUVEC-coated beads were imbedded in the fibrin clot per well of a96-well tissue culture plate. Conditioned medium derived from normalhuman fibroblasts (NHLF, Lonza) at 80%/o confluence was plated on top ofthe gel. DLL4 antibody and control antibody KLH at 15 μg/ml were addedonto the well. At day 10 and 12, images were taken with invertedmicroscope and Nikon CCD camera. DLL4 inhibition with E9 and A10antibodys results in enhancement of endothelial cell sprouting in vitro(data not shown).

Example 11: DLL4 Antibody Treatment Inhibited Tumor Growth In Vivo

The effect of anti-DLL4 antibodies on tumor growth was evaluated onsubcutaneous Calu-6 xenograft tumors implanted in SCID-Beige mice.Briefly, 2×10⁶ cells were inoculated subcutaneously into the right hindflank of female SCID-Beige mice. Tumors were allowed to establish for14-18 days, at which point tumor volume was determined using electroniccaliper measurements. Tumor size was calculated using the formula:L×W²/2. Mice were allocated into treatment groups (n=10 per group) sothat each cohort of animals had equivalent mean tumor volume prior toinitiation of therapy (typically between 180 and 250 mm³). Animals werethen dosed intraperitoneally twice a week for two weeks (total of 4doses) with anti-DLL4 antibodies. Tumor volume was measured on averagetwice a week for the duration of the experiment until the mean tumorvolume in each group reached an endpoint of ≥2,000 mm³. Results areshown in Table 31. For E9 series of PROfusion antibodies, those withimproved pharmacokinetics (as shown in Example 9) tend to have strongeranti-tumor activity in vivo.

TABLE 31 Efficacy of anti-DLL4 antibodies in the Calu-6 human non-smallcell lung cancer xenograft model. Treatment Dose Route, Regimen % T/Ca %ILSb E9 10 mg/kg IP, 2X/week X2 43** 52** E9-10C 10 mg/kg IP, 2X/week X226** 81** E9-2B 10 mg/kg IP, 2X/week X2 28** 76** E9-10E 10 mg/kg IP,2X/week X2 30** 70** E9-19 10 mg/kg IP, 2X/week X2 31** 64** E9-5E 10mg/kg IP, 2X/week X2 10** 57** E9-71 10 mg/kg IP, 2X/week X2 34** 63**E9-1F 10 mg/kg IP, 2X/week X2 34** 57** E9-12B 10 mg/kg IP, 2X/week X238** 52** E9-7E 10 mg/kg IP, 2X/week X2 38** 44** E9-10H 10 mg/kg IP,2X/week X2 41** 52** E9-66 10 mg/kg IP, 2X/week X2 43** 32*  a% T/C =mean tumor volume of treatment group/tumor volume of treatment controlgroup × 100. P values (as indicated by asterisks) are derived fromStudent's T test comparison of treatment group vs. treatment controlgroup. Based on day 25/26/27 measurements. b% ILS = (T − C)/C × 100,where T = median time to endpoint of treatment group and C = median timeto endpoint of treatment control group. P values (as indicated byasterisks) derived from Kaplan Meier log-rank comparison of treatmentgroup vs. treatment control group. Based on an endpoint of 2000 mm³. *p< 0.05; **p < 0.01

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

What is claimed is:
 1. A binding protein comprising an antigen bindingdomain capable of binding human DLL4, said antigen binding domaincomprising at least one or more CDRs selected from the group consistingof: CDR-H1: (SEQ ID NO: 99) X₁-X₂-X₃-X₄-X₅-X₆-X₇,

wherein; X₁ is S or N; X₂ is S, G, or N; X₃ is S, N, T, G, or R; X₄ isY; X₅ is Y or H; X₆ is W; and X₇ is G; CDR-H2: (SEQ ID NO: 100)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆,

wherein; X₁ is D; X₂ is I; X₃ is Y, N, or S; X₄ is Y; X₅ is T, N, A, I,S, or R; X₆ is G; X₇ is S, N, T, or G; X₈ is T; X₉ is Y; X₁₀ is Y; X₁₁is N; X₁₂ is P; X₁₃ is S; X₁₄ is L; X₁₅ is K; and X₁₆ is S, N, D, or G;CDR-H3: (SEQ ID NO: 101) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁,

wherein; X₁ is E, Y, F, Q, W, L, or A; X₂ is D, A, S, G, V, E, or N; X₃is V, M, L, P, or A; X₄ is I, A, P, R, S, K, Q, V, G, M, or E; X₅ is L,Y, F, or M; X₆ is R, G, S, Q, or A; X₇ is G; X₈ is G, A, or S; X₉ is S,A, L, V, R, or G; X₁₀ is D; and X₁₁ is Y, D, S, N, H, E, R, L, P, C, I,M, T, Q, or K; CDR-L1: (SEQ ID NO: 102)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁,

wherein; X₁ is S; X₂ is G; X₃ is Q, E, or D; X₄ is R, S, G, M, K, L, orT; X₅ is L; X₆ is G; X₇ is D or E; X₈ is K; X₉ is Y; X₁₀ is A or V; andX₁₁ is S; CDR-L2: (SEQ ID NO: 103) X₁-X₂-X₃-X₄-X₅-X₆-X₇,

wherein; X₁ is E or Q; X₂ is D; X₃ is S, L, T, A, E, or F; X₄ is K, T,E, N, Q, S, or M; X₅ is R; X₆ is P; and X₇ is S; and CDR-L3:(SEQ ID NO: 104) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉,

wherein; X₁ is Q; X₂ is A; X₃ is W; X₄ is D; X₅ is R, S, M, E, N, G, orK; X₆ is D or E; X₇ is T, V, A, S, or M; X₈ is G, A, or C; and X₉ is V.2. The binding protein according to claim 1, wherein said at least oneCDR comprises an amino acid sequence selected from the group consistingof: residues 31-37 of SEQ ID NO:1 (CDR-H1); residues 52-67 of SEQ IDNO:1 (CDR-H2); residues 100-110 of SEQ ID NO:1 (CDR-H3); residues 23-33of SEQ ID NO:111 (CDR-L1); residues 49-55 of SEQ ID NO:111 (CDR-L2);residues 88-96 of SEQ ID NO:111 (CDR-L3); SEQ ID NO:117 (CDR-H1); SEQ IDNO:118 (CDR-H2); SEQ ID NO:119 (CDR-H3); SEQ ID NO: 121 (CDR-H1); SEQ IDNO: 122 (CDR-H2); SEQ ID NO: 123 (CDR-H3); SEQ ID NO:125 (CDR-H1); SEQID NO:126 (CDR-H2); SEQ ID NO:127 (CDR-H3); SEQ ID NO: 129 (CDR-H1); SEQID NO: 130 (CDR-H2); SEQ ID NO: 131 (CDR-H3); SEQ ID NO:133 (CDR-H1);SEQ ID NO:134 (CDR-H2); SEQ ID NO:135 (CDR-H3); SEQ ID NO:137 (CDR-H1);SEQ ID NO:138 (CDR-H2); SEQ ID NO:139 (CDR-H3); SEQ ID NO:141 (CDR-H1);SEQ ID NO:142 (CDR-H2); SEQ ID NO:143 (CDR-H3); SEQ ID NO:145 (CDR-H1);SEQ ID NO:146 (CDR-H2); SEQ ID NO:147 (CDR-H3); SEQ ID NO:149 (CDR-H1);SEQ ID NO:150 (CDR-H2); SEQ ID NO:151 (CDR-H3); SEQ ID NO:153 (CDR-H1);SEQ ID NO:154 (CDR-H2); SEQ ID NO:155 (CDR-H3); SEQ ID NO: 157 (CDR-H1);SEQ ID NO: 158 (CDR-H2); SEQ ID NO:159 (CDR-H3); SEQ ID NO:161 (CDR-H1);SEQ ID NO:162 (CDR-H2); SEQ ID NO:163 (CDR-H3); SEQ ID NO:165 (CDR-H1);SEQ ID NO:166 (CDR-H2); SEQ ID NO:167 (CDR-H3); SEQ ID NO: 169 (CDR-H1);SEQ ID NO: 170 (CDR-H2); SEQ ID NO: 171 (CDR-H3); SEQ ID NO: 173(CDR-H1); SEQ ID NO: 174 (CDR-H2); SEQ ID NO: 175 (CDR-H3); SEQ ID NO:177 (CDR-H1); SEQ ID NO: 178 (CDR-H2); SEQ ID NO: 179 (CDR-H3); SEQ IDNO: 181 (CDR-H1); SEQ ID NO:182 (CDR-H2); SEQ ID NO:183 (CDR-H3); SEQ IDNO:185 (CDR-H1); SEQ ID NO:186 (CDR-H2); SEQ ID NO:187 (CDR-H3); SEQ IDNO: 189 (CDR-H1); SEQ ID NO: 190 (CDR-H2); SEQ ID NO: 191 (CDR-H3); SEQID NO:193 (CDR-H1); SEQ ID NO:194 (CDR-H2); SEQ ID NO:195 (CDR-H3); SEQID NO: 197 (CDR-H1); SEQ ID NO: 198 (CDR-H2); SEQ ID NO:199 (CDR-H3);SEQ ID NO:201 (CDR-H1); SEQ ID NO:202 (CDR-H2); SEQ ID NO:203 (CDR-H3);SEQ ID NO:205 (CDR-H1); SEQ ID NO:206 (CDR-H2); SEQ ID NO:207 (CDR-H3);SEQ ID NO:209 (CDR-H1); SEQ ID NO:210 (CDR-H2); SEQ ID NO:211 (CDR-H3);SEQ ID NO:213 (CDR-H1); SEQ ID NO:214 (CDR-H2); SEQ ID NO:215 (CDR-H3);SEQ ID NO:217 (CDR-L1); SEQ ID NO:218 (CDR-L2); SEQ ID NO:219 (CDR-L3);SEQ ID NO:221 (CDR-L1); SEQ ID NO:222 (CDR-L2); SEQ ID NO:223 (CDR-L3);SEQ ID NO:225 (CDR-L); SEQ ID NO:226 (CDR-L2); SEQ ID NO:227 (CDR-L3);SEQ ID NO:229 (CDR-L1): SEQ ID NO:230 (CDR-L2); SEQ ID NO:231 (CDR-L3);SEQ ID NO:233 (CDR-L); SEQ ID NO:234 (CDR-L2); SEQ ID NO:235 (CDR-L3);SEQ ID NO:237 (CDR-L1); SEQ ID NO:238 (CDR-L2); SEQ ID NO:239 (CDR-L3);SEQ ID NO:241 (CDR-L1): SEQ ID NO:242 (CDR-L2); SEQ ID NO:243 (CDR-L3);SEQ ID NO:245 (CDR-L1): SEQ ID NO:246 (CDR-L2); SEQ ID NO:247 (CDR-L3);SEQ ID NO:249 (CDR-L1); SEQ ID NO:250 (CDR-L2); SEQ ID NO:251 (CDR-L3);SEQ ID NO:253 (CDR-L): SEQ ID NO:254 (CDR-L2); SEQ ID NO:255 (CDR-L3);SEQ ID NO:257 (CDR-L1): SEQ ID NO:258 (CDR-L2); SEQ ID NO:259 (CDR-L3);SEQ ID NO:261 (CDR-L1); SEQ ID NO:262 (CDR-L2); SEQ ID NO:263 (CDR-L3);SEQ ID NO:265 (CDR-L); SEQ ID NO:266 (CDR-L2); SEQ ID NO:267 (CDR-L3);SEQ ID NO:269 (CDR-L1): SEQ ID NO:270 (CDR-L2); SEQ ID NO:271 (CDR-L3);SEQ ID NO:273 (CDR-L); SEQ ID NO:274 (CDR-L2); SEQ ID NO:275 (CDR-L3);SEQ ID NO:277 (CDR-L1); SEQ ID NO:278 (CDR-L2); SEQ ID NO:279 (CDR-L3);SEQ ID NO:281 (CDR-L1): SEQ ID NO:282 (CDR-L2); SEQ ID NO:283 (CDR-L3);SEQ ID NO:285 (CDR-L1): SEQ ID NO:286 (CDR-L2); SEQ ID NO:287 (CDR-L3);SEQ ID NO:289 (CDR-L1); SEQ ID NO:290 (CDR-L2); SEQ ID NO:291 (CDR-L3);SEQ ID NO:293 (CDR-L): SEQ ID NO:294 (CDR-L2); SEQ ID NO:295 (CDR-L3);SEQ ID NO:297 (CDR-L1): SEQ ID NO:298 (CDR-L2); SEQ ID NO:299 (CDR-L3);SEQ ID NO:301 (CDR-L); SEQ ID NO:302 (CDR-L2); SEQ ID NO:303 (CDR-L3);SEQ ID NO:305 (CDR-L); SEQ ID NO:306 (CDR-L2); SEQ ID NO:307 (CDR-L3);SEQ ID NO:309 (CDR-L1): SEQ ID NO:310 (CDR-L2); SEQ ID NO:311 (CDR-L3);SEQ ID NO:313 (CDR-L); SEQ ID NO:314 (CDR-L2); SEQ ID NO:315 (CDR-L3);residues 31-37 of SEQ ID NO:334 (CDR-H1); residues 52-67 of SEQ IDNO:334 (CDR-H2); residues 100-110 of SEQ ID NO:334 (CDR-H3); residues23-33 of SEQ ID NO:335 (CDR-L1); residues 49-55 of SEQ ID NO:335(CDR-L2); residues 88-96 of SEQ ID NO:335 (CDR-L3); residues 31-37 ofSEQ ID NO:336 (CDR-H1); residues 52-67 of SEQ ID NO:336 (CDR-H2);residues 100-110 of SEQ ID NO:336 (CDR-H3); residues 23-33 of SEQ IDNO:337 (CDR-L1); residues 49-55 of SEQ ID NO:337 (CDR-L2); residues88-96 of SEQ ID NO:337 (CDR-L3); residues 31-37 of SEQ ID NO:338(CDR-H1); residues 52-67 of SEQ ID NO:338 (CDR-H2); residues 100-110 ofSEQ ID NO:338 (CDR-H3); residues 23-33 of SEQ ID NO:339 (CDR-L1);residues 49-55 of SEQ ID NO:339 (CDR-L2); residues 88-96 of SEQ IDNO:339 (CDR-L3); residues 31-37 of SEQ ID NO:340 (CDR-H1); residues52-67 of SEQ ID NO:340 (CDR-H2); residues 100-110 of SEQ ID NO:340(CDR-H3); residues 23-33 of SEQ ID NO:341 (CDR-L1); residues 49-55 ofSEQ ID NO:341 (CDR-L2); residues 88-96 of SEQ ID NO:341 (CDR-L3);residues 31-37 of SEQ ID NO:342 (CDR-H1); residues 52-67 of SEQ IDNO:342 (CDR-H2); residues 100-110 of SEQ ID NO:342 (CDR-H3); residues23-33 of SEQ ID NO:343 (CDR-L1); residues 49-55 of SEQ ID NO:343(CDR-L2); residues 88-96 of SEQ ID NO:343 (CDR-L3); residues 31-37 ofSEQ ID NO:344 (CDR-H1); residues 52-67 of SEQ ID NO:344 (CDR-H2);residues 100-110 of SEQ ID NO:344 (CDR-H3); residues 24-34 of SEQ IDNO:345 (CDR-L1); residues 50-56 of SEQ ID NO:345 (CDR-L2); residues89-97 of SEQ ID NO:345 (CDR-L3); residues 31-37 of SEQ ID NO:346(CDR-H1); residues 52-67 of SEQ ID NO:346 (CDR-H2); residues 100-110 ofSEQ ID NO:346 (CDR-H3); residues 23-33 of SEQ ID NO:347 (CDR-L1);residues 49-55 of SEQ ID NO:347 (CDR-L2); residues 88-96 of SEQ IDNO:347 (CDR-L3); residues 31-37 of SEQ ID NO:348 (CDR-H1); residues52-67 of SEQ ID NO:348 (CDR-H2); residues 100-110 of SEQ ID NO:348(CDR-H3); residues 24-34 of SEQ ID NO:349 (CDR-L1); residues 50-56 ofSEQ ID NO:349 (CDR-L2); residues 89-97 of SEQ ID NO:349 (CDR-L3);residues 31-37 of SEQ ID NO:350 (CDR-H1); residues 52-67 of SEQ IDNO:350 (CDR-H2); residues 100-110 of SEQ ID NO:350 (CDR-H3); residues24-34 of SEQ ID NO:351 (CDR-L1); residues 50-56 of SEQ ID NO:351(CDR-L2); residues 89-97 of SEQ ID NO:351 (CDR-L3); residues 31-37 ofSEQ ID NO:352 (CDR-H1); residues 52-67 of SEQ ID NO:352 (CDR-H2);residues 100-110 of SEQ ID NO:352 (CDR-H3); residues 24-34 of SEQ IDNO:353 (CDR-L1); residues 50-56 of SEQ ID NO:353 (CDR-L2); residues89-97 of SEQ ID NO:353 (CDR-L3); residues 31-37 of SEQ ID NO:354(CDR-H1); residues 52-67 of SEQ ID NO:354 (CDR-H2); residues 100-110 ofSEQ ID NO:354 (CDR-H3); residues 24-34 of SEQ ID NO:355 (CDR-L1);residues 50-56 of SEQ ID NO:355 (CDR-L2); residues 89-97 of SEQ IDNO:355 (CDR-L3); residues 31-37 of SEQ ID NO:356 (CDR-H1); residues52-67 of SEQ ID NO:356 (CDR-H2); residues 100-110 of SEQ ID NO:356(CDR-H3); residues 23-33 of SEQ ID NO:357 (CDR-L1); residues 49-55 ofSEQ ID NO:357 (CDR-L2); residues 88-96 of SEQ ID NO:357 (CDR-L3);residues 31-37 of SEQ ID NO:358 (CDR-H1); residues 52-67 of SEQ IDNO:358 (CDR-H2); residues 100-110 of SEQ ID NO:358 (CDR-H3); residues23-33 of SEQ ID NO:359 (CDR-L1); residues 49-55 of SEQ ID NO:359(CDR-L2); residues 88-96 of SEQ ID NO:359 (CDR-L3); residues 31-37 ofSEQ ID NO:360 (CDR-H1); residues 52-67 of SEQ ID NO:360 (CDR-H2);residues 100-110 of SEQ ID NO:360 (CDR-H3); residues 23-33 of SEQ IDNO:361 (CDR-L1); residues 49-55 of SEQ ID NO:361 (CDR-L2); residues88-96 of SEQ ID NO:361 (CDR-L3).
 3. The binding protein according toclaim 2, wherein said binding protein comprises at least 3 CDRs.
 4. Thebinding protein according to claim 3, wherein said at least 3 CDRscomprises a variable domain CDR set selected from the group consistingof: VH E9 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:1 CDR-H2: residues52-67 of SEQ ID NO:1 CDR-H3 residues 100-110 of SEQ ID NO:1 VL E9 CDRSet CDR-L1: residues 23-33 of SEQ ID NO:111 CDR-L2: residues 49-55 ofSEQ ID NO:111 CDR-L3: residues 88-96 of SEQ ID NO:111 VH E9.4 CDR SetCDR-H1: SEQ ID NO:117 CDR-H2: SEQ ID NO: 118 CDR-H3: SEQ ID NO: 119 VLE9.4 CDR Set CDR-L1: SEQ ID NO:229 CDR-L2: SEQ ID NO:230 CDR-L3: SEQ IDNO:231 VH E9.11 CDR Set CDR-H1: SEQ ID NO:121 CDR-H2: SEQ ID NO: 122CDR-H3: SEQ ID NO:123 VL E9.11 CDR Set CDR-L1: SEQ ID NO:233 CDR-L2: SEQID NO:234 CDR-L3: SEQ ID NO:235 VH E9.14 CDR Set CDR-H1: SEQ ID NO: 125CDR-H2: SEQ ID NO: 126 CDR-H3: SEQ ID NO: 127 VL E9.14 CDR Set CDR-L1:SEQ ID NO:237 CDR-L2: SEQ ID NO:238 CDR-L3: SEQ ID NO:239 VH E9.17 CDRSet CDR-H1: SEQ ID NO: 129 CDR-H2: SEQ ID NO: 130 CDR-H3: SEQ ID NO: 131VL E9.17 CDR Set CDR-L1: SEQ ID NO:241 CDR-L2: SEQ ID NO:242 CDR-L3: SEQID NO:243 VH E9.18 CDR Set CDR-H1: SEQ ID NO:133 CDR-H2: SEQ ID NO: 134CDR-H3: SEQ ID NO: 135 VL E9.18 CDR Set CDR-L1: SEQ ID NO:245 CDR-L2:SEQ ID NO: 246 CDR-L3: SEQ ID NO:247 VH E9.19 CDR Set CDR-H1 SEQ IDNO:137 CDR-H2: SEQ ID NO: 138 CDR-H3: SEQ ID NO:139 VL E9.19 CDR SetCDR-L1: SEQ ID NO:249 CDR-L2: SEQ ID NO:250 CDR-L3: SEQ ID NO:251 VHE9.22 CDR Set CDR-H1: SEQ ID NO:141 CDR-H2: SEQ ID NO:142 CDR-H3: SEQ IDNO: 143 VL E9.22 CDR Set CDR-L1: SEQ ID NO:253 CDR-L2: SEQ ID NO:254CDR-L3: SEQ ID NO:255 VH E9.48 CDR Set CDR-H1: SEQ ID NO: 145 CDR-H2:SEQ ID NO: 146 CDR-H3: SEQ ID NO:147 VL E9.48 CDR Set CDR-L1: SEQ IDNO:257 CDR-L2: SEQ ID NO:258 CDR-L3: SEQ ID NO:259 VH E9.65 CDR SetCDR-H1: SEQ ID NO: 149 CDR-H2: SEQ ID NO:150 CDR-H3: SEQ ID NO:151 VLE9.65 CDR Set CDR-L1: SEQ ID NO:261 CDR-L2: SEQ ID NO:262 CDR-L3: SEQ IDNO: 263 VH E9.66 CDR Set CDR-H1: SEQ ID NO:153 CDR-H2: SEQ ID NO: 154CDR-H3: SEQ ID NO: 155 VL E9.66 CDR Set CDR-L1: SEQ ID NO:265 CDR-L2:SEQ ID NO:266 CDR-L3: SEQ ID NO:267 VH E9.71 CDR Set CDR-H1: SEQ IDNO:157 CDR-H2: SEQ ID NO: 158 CDR-H3: SEQ ID NO:159 VL E9.71 CDR SetCDR-L1: SEQ ID NO:269 CDR-L2: SEQ ID NO:270 CDR-L3: SEQ ID NO:271 VHE9.13 CDR Set CDR-H1: SEQ ID NO:161 CDR-H2: SEQ ID NO: 162 CDR-H3: SEQID NO: 163 VL E9.13 CDR Set CDR-L1: SEQ ID NO:217 CDR-L2: SEQ ID NO:218CDR-L3: SEQ ID NO:219 VH E9.16 CDR Set CDR-H1: SEQ ID NO: 165 CDR-H2:SEQ ID NO: 166 CDR-H3: SEQ ID NO: 167 VL E9.16 CDR Set CDR-L1: SEQ IDNO:221 CDR-L2: SEQ ID NO:222 CDR-L3: SEQ ID NO:223 VH E9.38 CDR SetCDR-H1: SEQ ID NO:169 CDR-H2: SEQ ID NO:170 CDR-H3: SEQ ID NO: 171 VLE9.38 CDR Set CDR-L1: SEQ ID NO:225 CDR-L2: SEQ ID NO:226 CDR-L3: SEQ IDNO:227 VH E9.2B CDR Set CDR-H1: SEQ ID NO:173 CDR-H2: SEQ ID NO: 174CDR-H3: SEQ ID NO:175 VL E9.2B CDR Set CDR-L1: SEQ ID NO:273 CDR-L2: SEQID NO:274 CDR-L3: SEQ ID NO:275 VH E9.1F CDR Set CDR-H1: SEQ ID NO:177CDR-H2: SEQ ID NO:178 CDR-H3: SEQ ID NO:179 VL E9.1F CDR Set CDR-L1: SEQID NO:277 CDR-L2: SEQ ID NO:278 CDR-L3: SEQ ID NO:279 VH E9.10H CDR SetCDR-H1: SEQ ID NO:181 CDR-H2: SEQ ID NO: 182 CDR-H3: SEQ ID NO:183 VLE9.10H CDR Set CDR-L1: SEQ ID NO:301 CDR-L2: SEQ ID NO:302 CDR-L3: SEQID NO:303 VH E9.5E CDR Set CDR-H1: SEQ ID NO:185 CDR-H2: SEQ ID NO:186CDR-H3: SEQ ID NO: 187 VL E9.5E CDR Set CDR-L1: SEQ ID NO:293 CDR-L2:SEQ ID NO:294 CDR-L3: SEQ ID NO: 295 VH E9.10C CDR Set CDR-H1: SEQ IDNO: 189 CDR-H2: SEQ ID NO: 190 CDR-H3: SEQ ID NO: 191 VL E9.10C CDR SetCDR-L1: SEQ ID NO:281 CDR-L2: SEQ ID NO:282 CDR-L3: SEQ ID NO:283 VHE9.7E CDR Set CDR-H1: SEQ ID NO:193 CDR-H2: SEQ ID NO: 194 CDR-H3: SEQID NO: 195 VL E9.7E CDR Set CDR-L1: SEQ ID NO:289 CDR-L2: SEQ ID NO:290CDR-L3: SEQ ID NO:291 VH E9.12B CDR Set CDR-H1: SEQ ID NO: 197 CDR-H2:SEQ ID NO: 198 CDR-H3: SEQ ID NO: 199 VL E9.12B CDR Set CDR-L1: SEQ IDNO:297 CDR-L2: SEQ ID NO:298 CDR-L3: SEQ ID NO:299 VH E9.10E CDR SetCDR-H1: SEQ ID NO:201 CDR-H2: SEQ ID NO:202 CDR-H3: SEQ ID NO:203 VLE9.10E CDR Set CDR-L1: SEQ ID NO:285 CDR-L2: SEQ ID NO:286 CDR-L3: SEQID NO:287 VH E9.6A CDR Set CDR-H1: SEQ ID NO:205 CDR-H2: SEQ ID NO:206CDR-H3: SEQ ID NO:207 VL E9.6A CDR Set CDR-L1: SEQ ID NO:305 CDR-L2: SEQID NO:306 CDR-L3: SEQ ID NO:307 VH E9.7A CDR Set CDR-H1: SEQ ID NO:209CDR-H2: SEQ ID NO:210 CDR-H3: SEQ ID NO:211 VL E9.7A CDR Set CDR-L1: SEQID NO:309 CDR-L2: SEQ ID NO:310 CDR-L3: SEQ ID NO:311 VH E9.8H CDR SetCDR-H1: SEQ ID NO:213 CDR-H2: SEQ ID NO:214 CDR-H3: SEQ ID NO:215 VLE9.8H CDR Set CDR-L1: SEQ ID NO:313 CDR-L2: SEQ ID NO:314 CDR-L3: SEQ IDNO:315 VH E9.1 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:334 CDR-H2:residues 52-67 of SEQ ID NO:334 CDR-H3: residues 100-110 of SEQ IDNO:334 VL E9.1 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:335 CDR-L2:residues 49-55 of SEQ ID NO:335 CDR-L3: residues 88-96 of SEQ ID NO:335VH E9-SE1 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:336 CDR-H2:residues 52-67 of SEQ ID NO:336 CDR-H3: residues 100-110 of SEQ IDNO:336 VL E9-SE CDR Set CDR-L: residues 23-33 of SEQ ID NO:337 CDR-L2:residues 49-55 of SEQ ID NO:337 CDR-L3: residues 88-96 of SEQ ID NO:337VH E9-SE2 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:338 CDR-H2:residues 52-67 of SEQ ID NO:338 CDR-H3: residues 100-110 of SEQ IDNO:338 VL E9-SE2 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:339 CDR-L2:residues 49-55 of SEQ ID NO:339 CDR-L3: residues 88-96 of SEQ ID NO:339VH E9-SE3 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:340 CDR-H2:residues 52-67 of SEQ ID NO:340 CDR-H3: residues 100-110 of SEQ IDNO:340 VL E9-SE3 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:341 CDR-L2:residues 49-55 of SEQ ID NO:341 CDR-L3: residues 88-96 of SEQ ID NO:341VH E9-SE4 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:342 CDR-H2:residues 52-67 of SEQ ID NO:342 CDR-H3: residues 100-110 of SEQ IDNO:342 VL E9-SE4 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:343 CDR-L2:residues 49-55 of SEQ ID NO:343 CDR-L3: residues 88-96 of SEQ ID NO:343VH E9-SE5 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:344 CDR-H2:residues 52-67 of SEQ ID NO:344 CDR-H3: residues 100-110 of SEQ IDNO:344 VL E9-SE5 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:345 CDR-L2:residues 50-56 of SEQ ID NO:345 CDR-L3: residues 89-97 of SEQ ID NO:345VH E9-SE6 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:346 CDR-H2:residues 52-67 of SEQ ID NO:346 CDR-H3: residues 100-110 of SEQ IDNO:346 VL E9-SE6 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:347 CDR-L2:residues 49-55 of SEQ ID NO:347 CDR-L3: residues 88-96 of SEQ ID NO:347VH E9-SE7 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:348 CDR-H2:residues 52-67 of SEQ ID NO:348 CDR-H3: residues 100-110 of SEQ IDNO:348 VL E9-SE7 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:349 CDR-L2:residues 50-56 of SEQ ID NO:349 CDR-L3: residues 89-97 of SEQ ID NO:349VH E9-SE8 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:350 CDR-H2:residues 52-67 of SEQ ID NO:350 CDR-H3: residues 100-110 of SEQ IDNO:350 VL E9-SE8 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:351 CDR-L2:residues 50-56 of SEQ ID NO:351 CDR-L3: residues 89-97 of SEQ ID NO:351VH E9-FR1 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:352 CDR-H2:residues 52-67 of SEQ ID NO:352 CDR-H3: residues 100-110 of SEQ IDNO:352 VL E9-FR1 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:353 CDR-L2:residues 50-56 of SEQ ID NO:353 CDR-L3: residues 89-97 of SEQ ID NO:353VH E9-FR2 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:354 CDR-H2:residues 52-67 of SEQ ID NO:354 CDR-H3: residues 100-110 of SEQ IDNO:354 VL E9-FR2 CDR Set CDR-L1: residues 24-34 of SEQ ID NO:355 CDR-L2:residues 50-56 of SEQ ID NO:355 CDR-L3: residues 89-97 of SEQ ID NO:355VH E9.71 CDR Set CDR-H1: residues 31-37 of SEQ ID NO:356 CDR-H2:residues 52-67 of SEQ ID NO:356 CDR-H3: residues 100-110 of SEQ IDNO:356 VL E9.71 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:357 CDR-L2:residues 49-55 of SEQ ID NO:357 CDR-L3: residues 88-96 of SEQ ID NO:357VH E9.71(M) CDR Set CDR-H1: residues 31-37 of SEQ ID NO:358 CDR-H2:residues 52-67 of SEQ ID NO:358 CDR-H3: residues 100-110 of SEQ IDNO:358 VL E9.71(M) CDR Set CDR-L1: residues 23-33 of SEQ ID NO:359CDR-L2: residues 49-55 of SEQ ID NO:359 CDR-L3: residues 88-96 of SEQ IDNO:359 VH E9.71(L) CDR Set CDR-H1: residues 31-37 of SEQ ID NO:360CDR-H2: residues 52-67 of SEQ ID NO:360 CDR-H3: residues 100-110 of SEQID NO:360 VL E9.71(L) CDR Set CDR-L1: residues 23-33 of SEQ ID NO:361CDR-L2: residues 49-55 of SEQ ID NO:361 CDR-L3: residues 88-96 of SEQ IDNO:361
 5. The binding protein according to claim 4, comprising at leasttwo variable domain CDR sets.
 6. The binding protein according to claim5, wherein said at least two variable domain CDR sets are selected froma group consisting of: VH E9 CDR Set and VL E9 CDR Set, VH E9.4 CDR Setand VL E9.4 CDR Set, VH E9.11 CDR Set and VL E9.11 CDR Set, VH E9.14 CDRSet and VL E9.14 CDR Set, VH E9.17 CDR Set and VL E9.17 CDR Set, VHE9.18 CDR Set and VL E9.18 CDR Set, VH E9.19 CDR Set and VL E9.19 CDRSet, VH E9.22 CDR Set and VL E9.22 CDR Set, VH E9.48 CDR Set and VLE9.48 CDR Set, VH E9.65 CDR Set and VL E9.65 CDR Set, VH E9.66 CDR Setand VL E9.66 CDR Set, VH E9.71 CDR Set and VL E9.71 CDR Set, VH E9.13CDR Set and VL E9.13 CDR Set, VH E9.16 CDR Set and VL E9.16 CDR Set, VHE9.38 CDR Set and VL E9.38 CDR Set, VH E9.2B CDR Set and VL E9.2B CDRSet, VH E9.1F CDR Set and VL E9.1F CDR Set, VH E9.10H CDR Set and VLE9.10H CDR Set, VH E9.5E CDR Set and VL E9.5E CDR Set, VH E9.10C CDR Setand VL E9.10C CDR Set VH E9.7E CDR Set and VL E9.7E CDR Set, VH E9.12BCDR Set and VL E9.12B CDR Set, VH E9.10E CDR Set and VL E9.10E CDR Set,VH E9.6A CDR Set and VL E9.6A CDR Set, VH E9.7A CDR Set and VL E9.7A CDRSet, VH E9.8H CDR Set and VL E9.8H CDR Set, VH E9-SE1 CDR Set and VLE9-SE1 CDR Set, VH E9-SE2 CDR Set and VL E9-SE2 CDR Set, VH E9-SE3 CDRSet and VL E9-SE3 CDR Set, VH E9-SE4 CDR Set and VL E9-SE4 CDR Set VHE9-SE5 CDR Set and VL E9-SE5 CDR Set, VH E9-SE6 CDR Set and VL E9-SE6CDR Set, VH E9-SE7 CDR Set and VL E9-SE7 CDR Set, VH E9-SE8 CDR Set andVL E9-SE8 CDR Set, VH E9-FR1 CDR Set and VL E9-FR1 CDR Set VH E9-FR2 CDRSet and VL E9-FR2 CDR Set, VH E9.71 CDR Set and VL E9.71 CDR Set, VHE9.71(M) CDR Set and VL E9.71(M) CDR Set, and VH E9.71(L) CDR Set and VLE9.71(L) CDR Set.
 7. The binding protein according to any one of claims1-6, further comprising a human acceptor framework.
 8. The bindingprotein according to claim 7, wherein said human acceptor frameworkcomprises an amino acid sequence selected from the group consisting of:heavy chain acceptor framework sequences SEQ ID NOS:6-22, heavy chainacceptor sequences SEQ ID NOS:35-62, light chain acceptor sequences SEQID NOS:23-34, and light chain acceptor sequences SEQ ID NOS:63-98. 9.The binding protein according to claim 7 or claim 8, wherein said humanacceptor framework comprises at least one framework region amino acidsubstitution, wherein the amino acid sequence of the framework is atleast 65% identical to the sequence of said human acceptor framework andcomprising at least 70 amino acid residues identical to said humanacceptor framework.
 10. The binding protein according to claim 8,wherein said human acceptor framework comprises at least one frameworkregion amino acid substitution at a key residue, said key residueselected from the group consisting of: a residue adjacent to a CDR; aglycosylation site residue; a rare residue; a residue capable ofinteracting with human DLL4 a canonical residue; a contact residuebetween heavy chain variable region and light chain variable region; aresidue within a Vernier zone; and a residue in a region that overlapsbetween a Chothia-defined variable heavy chain CDR1 and a Kabat-definedfirst heavy chain framework.
 11. The binding protein according to claim10, wherein key residue selected from the group consisting of: 2H, 4H,24H, 26H, 27H, 29H, 34H, 35H, 37H, 39H, 44H, 45H, 47H, 48H, 49H, 50H,51H, 58H, 59H, 60H, 63H, 67H, 69H, 71H, 73H, 76H, 78H, 91H, 93H, 94H,2L, 4L, 25L, 29L, 27bL, 33L, 34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L,49L, 55L, 58L, 62L, 64L, 71L, 87L, 89L, 90L, 91L, 94L, 95L.
 12. Thebinding protein according to claim 11, where the binding protein is aconsensus human variable domain.
 13. The binding protein according toclaim 1, where said binding protein comprises at least one variabledomain having amino acid sequence selected from the group consisting of:SEQ ID NOS:1, 111, 116, 228, 120, 232, 124, 236, 128, 240, 132, 244,136, 248, 140, 252, 144, 256, 148, 260, 152, 264, 156, 268, 160, 216,164, 220, 168, 224, 172, 272, 176, 276, 180, 300, 184, 292, 188, 280,192, 288, 196, 296, 200, 284, 204, 304, 208, 308, 212, 312, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, and
 361. 14. Thebinding protein according to claim 13, wherein said binding proteincomprises two variable domains, wherein said two variable domains haveamino acid sequences selected from the group consisting of: SEQ ID NOS:1 and 111, SEQ ID NOS: 116 and 228, SEQ ID NOS: 120 and 232, SEQ ID NOS:124 and 236, SEQ ID NOS: 128 and 240, SEQ ID NOS: 132 and 244, SEQ IDNOS: 136 and 248, SEQ ID NOS: 140 and 252, SEQ ID NOS:144 and 256, SEQID NOS:148 and 260, SEQ ID NOS:152 and 264, SEQ ID NOS:156 and 268, SEQID NOS: 160 and 216, SEQ ID NOS: 164 and 220, SEQ ID NOS: 168 and 224,SEQ ID NOS: 172 and 272, SEQ ID NOS: 176 and 276, SEQ ID NOS:180 and300, SEQ ID NOS:184 and 292, SEQ ID NOS:188 and 280, SEQ ID NOS: 192 and288, SEQ ID NOS: 196 and 296, SEQ ID NOS:200 and 284, SEQ ID NOS:204 and304, SEQ ID NOS:208 and 308, SEQ ID NOS:212 and 312, SEQ ID NOS:334 and335, SEQ ID NOS:336 and 337, SEQ ID NOS:338 and 339, SEQ ID NOS:340 and341, SEQ ID NOS:342 and 343, SEQ ID NOS:344 and 345, SEQ ID NOS:346 and347, SEQ ID NOS:348 and 349, SEQ ID NOS:350 and 351, SEQ ID NOS:352 and353, SEQ ID NOS:354 and 355, SEQ ID NOS:356 and 357, SEQ ID NOS:358 and359, SEQ ID NOS:360 and
 361. 15. The binding protein according to claim11, wherein said binding protein comprises at least one variable domainhaving an amino acid sequence selected from the group consisting of: SEQID NOS:1, 111, 116, 228, 120, 232, 124, 236, 128, 240, 132, 244, 136,248, 140, 252, 144, 256, 148, 260, 152, 264, 156, 268, 160, 216, 164,220, 168, 224, 172, 272, 176, 276, 180, 300, 184, 292, 188, 280, 192,288, 196, 296, 200, 284, 204, 304, 208, 308, 212, 312, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, and
 361. 16. Thebinding protein according to claim 2 wherein said antigen binding domaincomprises a V_(H).
 17. The binding protein according to claim 16 whereinsaid V_(H) comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NOS:1, 116, 120, 124, 128, 132, 136, 140, 144,148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200,204, 208, 212, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, and
 360. 18. The binding protein according to claim 2 whereinsaid antigen binding domain comprises a V_(L).
 19. The binding proteinaccording to claim 18 wherein said V_(L) comprises an amino acidsequence selected from the group consisting of: SEQ ID NOS:111, 228,232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 216, 220, 224, 272,276, 300, 292, 280, 288, 296, 284, 304, 308, 312, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, and
 361. 20. The bindingprotein according to claim 2 wherein said antigen binding domaincomprises a V_(H) and a V_(L).
 21. The binding protein according toclaim 19 further comprising a V_(H) wherein said V_(H) comprises anamino acid sequence selected from the group consisting of: SEQ ID NOS:1,116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168,172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, and
 360. 22. Thebinding protein according to claim 20 wherein said V_(L) comprises anamino acid sequence selected from the group consisting of: SEQ ID NOS:111, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 216, 220,224, 272, 276, 300, 292, 280, 288, 296, 284, 304, 308, 312, 335, 337,339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, and
 361. 23. Thebinding protein according to claim 2, further comprising a heavy chainimmunoglobulin constant domain selected from the group consisting of: ahuman IgM constant domain; a human IgG1 constant domain; a human IgG2constant domain; a human IgG3 constant domain; a human IgG4 constantdomain; a human IgE constant domain and a human IgA constant domain. 24.The binding protein according to claim 23 wherein said heavy chainimmunoglobulin constant region domain is a human IgG1 constant domain.25. The binding protein according to claim 24 wherein said human IgG1constant domain comprises amino acid sequence selected from the groupconsisting of SEQ ID NO:2 and SEQ ID NO:3.
 26. The binding proteinaccording to claim 2, further comprising a light chain immunoglobulinconstant domain selected from the group consisting of: a human Ig kappaconstant domain and a human Ig lambda constant domain.
 27. The bindingprotein according to claim 26 wherein said light chain immunoglobulinconstant region domain is a human Ig kappa constant domain comprisingamino acid sequence SEQ ID NO:4.
 28. The binding protein according toclaim 26 wherein said light chain immunoglobulin constant region domainis a human Ig lambda constant domain comprising amino acid sequence SEQID NO:5.
 29. The binding protein according to claim 1 wherein saidbinding protein is selected from the group consisting of: animmunoglobulin molecule, an scFv, a monoclonal antibody, a humanantibody, a chimeric antibody, a humanized antibody, a single domainantibody, a Fab fragment, a Fab′ fragment, an F(ab′)₂, an Fv, adisulfide linked Fv, a single domain antibody, a diabody, amultispecific antibody, a bispecific antibody, and a dual specificantibody.
 30. The binding protein according to claim 29 wherein saidbinding protein is a human antibody.
 31. A binding protein capable ofbinding human DLL-4, said binding protein comprising: an Ig constantheavy region having an amino acid sequence selected from the groupconsisting of SEQ ID NO:2 and SEQ ID NO:3; an Ig constant light regionhaving an amino acid sequence selected from the group consisting of SEQID NO:4 and SEQ ID NO:5; an Ig variable heavy region having an aminoacid sequence selected from the group consisting: SEQ ID NOS:1, 116,120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172,176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 334, 336, 338, 340,342, 344, 346, 348, 350, 352, 354, 356, 358, and 360; and an Ig variablelight region having an amino acid sequence selected from the groupconsisting: SEQ ID NOS: 111, 228, 232, 236, 240, 244, 248, 252, 256,260, 264, 268, 216, 220, 224, 272, 276, 300, 292, 280, 288, 296, 284,304, 308, 312, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, and
 361. 32. The binding protein according to claim 31,wherein the Ig constant light region is SEQ ID NO:5.
 33. The bindingprotein according to any one of claims 1-32, wherein the binding proteinis capable of blocking DLL4 interaction with a Notch protein selectedfrom the group consisting of Notch-1, Notch-2, Notch-3, Notch-4, andcombinations thereof.
 34. The binding protein according to claim 33,wherein the binding protein is capable of blocking DLL4 interaction withNotch-1 and Notch-4.
 35. The binding protein according to claim 33,wherein the binding protein is capable of blocking DLL4 interaction withNotch-1.
 36. The binding protein according to claim 33, wherein thebinding protein is capable of blocking DLL4 interaction with Notch-4.37. The binding protein according to any one of claims 1-32, wherein thebinding protein is capable of modulating a biological function of DLL4.38. The binding protein according to any one of claims 1-32, whereinsaid binding protein is capable of neutralizing a DLL4.
 39. Theneutralizing binding protein according to claim 38 wherein said DLL4 isselected from the group consisting of: human DLL4, mouse DLL4,cynomolgus DLL4, and rat DLL4.
 40. The neutralizing binding proteinaccording to claim 38 wherein said neutralizing binding proteindiminishes the ability of DLL4 to bind to its receptor.
 41. Theneutralizing binding protein according to claim 38 wherein saidneutralizing binding protein is capable of reducing normal angiogenesis.42. The neutralizing binding protein according to claim 38, wherein saidneutralizing binding protein has a dissociation constant (K_(D))selected from the group consisting of; at most about 10⁻⁷ M; at mostabout 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at most about10⁻¹¹ M; at most about 10⁻¹² M; and at most 10⁻¹³ M.
 43. Theneutralizing binding protein according to claim 38, wherein saidneutralizing binding protein has an on rate selected from the groupconsisting of; at least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹s⁻¹; atleast about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about10⁶M⁻¹s⁻¹.
 44. The neutralizing binding protein according to claim 38,wherein said neutralizing binding protein has an off rate selected fromthe group consisting of; at most about 10⁻³s⁻¹; at most about 10⁻⁴s⁻¹;at most about 10⁻⁵s⁻¹; and at most about 10⁻⁶s⁻¹.
 45. A labeled bindingprotein comprising a binding protein of any one of claims 1-32, whereinsaid binding protein is conjugated to a detectable label.
 46. Thelabeled binding protein of claim 45, wherein the detectable label isselected from the group consisting of a radiolabel, an enzyme, afluorescent label, a luminescent label, a bioluminescent label, amagnetic label, and biotin.
 47. The labeled binding protein of claim 46,wherein said label is a radiolabel selected from the group consistingof: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm.48. An antibody construct comprising a binding protein described in anyof claims 1-32 and further comprising a linker polypeptide or animmunoglobulin constant domain.
 49. The antibody construct according toclaim 48, selected from the group consisting of: an immunoglobulinmolecules, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, a Fab, a Fab′, a F(ab′)₂, a Fv, adisulfide linked Fv, a scFv, a single domain antibody, a diabody, amultispecific antibody, a dual specific antibody, and a bispecificantibody.
 50. The antibody construct according to claim 48, wherein saidantibody construct comprises a heavy chain immunoglobulin constantdomain selected from the group consisting of: a human IgM constantdomain, a human IgG1 constant domain, a human IgG2 constant domain, ahuman IgG3 constant domain, a human IgG4 constant domain, a human IgEconstant domain, a human IgA constant domain, and a IgG constant domainvariant with one or more mutations altering binding strength to Fcneonatal receptor, Fc gamma receptors, or C1q.
 51. The antibodyconstruct according to claim 48, comprising an immunoglobulin constantdomain having an amino acid sequence selected from consisting of: SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinations thereof.52. An antibody conjugate comprising an antibody construct as describedin claim 48, wherein said antibody construct is conjugated to atherapeutic or cytotoxic agent.
 53. The antibody conjugate of claim 52,wherein said therapeutic or cytotoxic agent is selected from the groupconsisting of: an anti-metabolite, an alkylating agent, an antibiotic, agrowth factor, a cytokine, an anti-angiogenic agent, an anti-mitoticagent, an anthracycline, toxin, and an apoptotic agent.
 54. An isolatednucleic acid encoding a polypeptide selected from the group consistingof: a polypeptide comprising a heavy chain variable domain, wherein theheavy chain variable domain comprises one or more of CDR-H1, a CDR-H2,or a CDR-H3 as described in claim 1: a polypeptide comprising a lightchain variable domain, wherein the light chain variable domain comprisesone or more of CDR-L1, a CDR-L2, or a CDR-L3 as described in claim 1; ora combination of both polypeptides.
 55. A vector comprising an isolatednucleic acid according to claim
 54. 56. The vector of claim 55 whereinsaid vector is selected from the group consisting of: pcDNA, pTT, pTT3,pEFBOS, pBV, pJV, pHybE and pBJ.
 57. A host cell comprising a vectoraccording to any one of claims 55 and
 56. 58. The host cell according toclaim 57, wherein said host cell is a prokaryotic cell.
 59. The hostcell according to claim 58, wherein said host cell is Escherichia coli.60. The host cell according to claim 59, wherein said host cell is aeukaryotic cell.
 61. The host cell according to claim 60, wherein saideukaryotic cell is selected from the group consisting of: a protistcell, an animal cell, a plant cell, and a fungal cell.
 62. The host cellaccording to claim 61, wherein said eukaryotic cell is an animal cellselected from the group consisting of: a mammalian cell, an avian cell,and an insect cell.
 63. The host cell according to claim 62, whereinsaid mammalian cell is a CHO cell.
 64. The host cell according to claim62, wherein said mammalian cell is a COS cell.
 65. The host cellaccording to claim 61, wherein said fungal cell is Saccharomycescerevisiae.
 66. The host cell according to claim 62, wherein said insectcell is an Sf9 cell.
 67. A method of producing a binding protein thatbinds human DLL4, comprising culturing the host cell of any one ofclaims 57-66 in a culture medium under conditions sufficient to producea binding protein that binds human DLL4.
 68. A binding protein producedaccording to the method of claim
 67. 69. A crystallized binding proteincomprising a binding protein according to any one of claims 1-32,wherein said binding protein exists as a crystal.
 70. The crystallizedbinding protein according to claim 69, wherein said crystal is acarrier-free pharmaceutical controlled release crystal.
 71. Thecrystallized binding protein according to claim 69, wherein said bindingprotein has a greater half life in vivo than the soluble counterpart ofsaid binding protein.
 72. The crystallized binding protein according toclaim 69, wherein said binding protein retains biological activity. 73.A composition for the release of a binding protein said compositioncomprising: (a) a formulation, wherein said formulation comprises acrystallized binding protein, according to any one of claims 69-72, andan ingredient; and (b) at least one polymeric carrier.
 74. Thecomposition according to claim 73, wherein said polymeric carrier is apolymer selected from one or more of the group consisting of: poly(acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid),poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutyrate), poly(caprolactone), poly (dioxanone), poly (ethylene glycol), poly((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolyeaccharides, blends and copolymers thereof.
 75. The compositionaccording to claim 73, wherein said ingredient is selected from thegroup consisting of albumin, sucrose, trehalose, lactitol, gelatin,hydroxypropyl-f-cyclodextrin, methoxypolyethylene glycol andpolyethylene glycol.
 76. A method for treating a mammal comprising thestep of administering to the mammal an effective amount of thecomposition according to claim
 73. 77. A pharmaceutical compositioncomprising the binding protein of any one of claims 1-32, and apharmaceutically acceptable carrier.
 78. The pharmaceutical compositionof claim 77 which further comprises at least one additional therapeuticagent for treating a disorder in which DLL4 activity is detrimental. 79.The pharmaceutical composition of claim 78, wherein said additionalagent is selected from the group consisting of: angiogenesis inhibitors;kinase inhibitors; co-stimulation molecule blockers; adhesion moleculeblockers: anti-cytokine antibody or functional fragment thereof;methotrexate; corticosteroids; cyclosporine; rapamycin; FK506; andnon-steroidal anti-inflammatory agents.
 80. A method for reducing humanDLL4 activity comprising contacting human DLL4 with the binding proteinof any one of claims 1-32 such that human DLL4 activity is reduced. 81.A method for reducing human DLL4 activity in a human subject sufferingfrom a disorder in which DLL4 activity is detrimental, comprisingadministering to the human subject the binding protein of any one ofclaims 1-32 such that human IL-17 activity in the human subject isreduced.
 82. A method for treating a subject for a disease or a disorderin which DLL4 activity is detrimental by administering to the subjectthe binding protein of any one of claims 1-32 such that treatment isachieved.
 83. The method of claim 82, wherein said disorder is selectedfrom the group consisting of: breast cancer, colon cancer, rectalcancer, lung cancer, oropharynx cancer, hypopharynx cancer, esophagealcancer, stomach cancer, pancreas cancer, liver cancer, gallbladdercancer, bile duct cancer, small intestine cancer, urinary tract cancer,female genital tract cancer, male genital tract cancer, endocrine glandcancer, skin cancer, hemangiomas, melanomas, sarcomas, brain tumor,nerve cancer, eye tumor, meninges cancer, solid tumors fromhematopoietic malignancies, tumor metastases, ocular neovascularization,edema, rheumatoid arthritis, multiple sclerosis, atheroscleoroticplaques, Crohn's disease, inflammatory bowel disease, refractoryascites, psoriasis, sarcoidosis, arterial arteriosclerosis, sepsis,peptic ulcers, burns, and pancreatitis, polycystic ovarian disease(POD), endometriosis, uterine fibroids, benign prostate hypertrophy, andother angiogenesis independent and dependent diseases characterized byabberant DLL4 activity.
 84. The method according to claim 83, whereinthe disorder is a primary and metastatic cancer.
 85. The methodaccording to claim 83, wherein the urinary tract cancer is selected fromthe group consisting of renal cancer, bladder cancer, and urotheliumcancer.
 86. The method according to claim 83, wherein the female genitaltract cancer is selected from the group consisting of cervical cancer,uterine cancer, ovarian cancer, choriocarcinoma, and gestationaltrophoblastic disease.
 87. The method according to claim 83, wherein themale genital tract cancer is selected from the group consisting ofprostate cancer, seminal vesicles cancer, testicular cancer, and germcell tumor.
 88. The method according to claim 83, wherein the endocrinegland cancer is selected from the group consisting of thyroid cancer,adrenal cancer, and pituitary gland cancer.
 89. The method according toclaim 83, wherein the sarcoma is selected from the group consisting of abone sarcoma, a soft tissue sarcoma, and Kaposi's sarcoma.
 90. Themethod according to claim 83, wherein the meninges cancer is selectedfrom the group consisting of an astrocytoma, a glioma, a glioblastoma, aretinoblastoma, a neuroma, a neuroblastoma, a Schwannoma, and ameningiomas.
 91. The method according to claim 83, wherein the solidtumor from a hematopoietic malignancy is a leukemia, a Hodgkin'sleukemia, a non-Hodgkin's leukemia, a lymphoma, a Hodgkin's lymphoma,and a non-Hodgkin's lymphomas.
 92. The method according to claim 83,wherein the ocular neovascularization is selected from the groupconsisting of diabetic blindness, a retinopathy, an age-induced maculardegeneration, and a rubeosis.
 93. A method of treating a patientsuffering from a disorder in which DLL4 is detrimental comprising thestep of administering the binding protein of any one of claims 1-32before, concurrent with, or after the administration of a second agent,wherein the second agent is selected from the group consisting of anantibody or fragment thereof capable of binding human VEGFR2;methotrexate; an antibody, or fragment thereof, capable of binding humanTNF: corticosteroids, cyclosporine, rapamycin, FK506, and non-steroidalanti-inflammatory agents.
 94. A binding protein comprising an antigenbinding domain capable of binding human DLL4, said antigen bindingdomain comprising at least one or more CDRs selected from the groupconsisting of: CDR-H1: (SEQ ID NO: 105) X₁-X₂-X₃-X₄-X₅,

wherein; X₁ is S, N, or D; X₂ is H or Y; X₃ is W; X₄ is M; X₅ is S or H;CDR-H2: (SEQ ID NO: 106)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇,

wherein; X₁ is I, D, M, or T; X₂ is I; X₃ is S; X₄ is Y, N, S, Q, V, T,H, or D; X₅ is D; X₆ is G; X₇ is S, R, I, T, G, K, H, or N; X₈ is N, Y,S, I, or T; X₉ is K, M, N, Q, E, T, R, S, A, or L; X₁₀ is Y, D, or E;X₁₁ is S or Y; X₁₂ is A; X₁₃ is D; X₁₄ is S; X₁₅ is V; X₁₆ is K; and X₁₇is G; CDR-H3: (SEQ ID NO: 107) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀,

wherein; X₁ is A; X₂ is G, A, or R; X₃ is G; X₄ is G, S, or A; X₅ is N;X₆ is V or M; X₇ is G; X₈ is F, L, Y, or M; X₉ is D; and X₁₀ is I, S, orL; CDR-L1: (SEQ ID NO: 108) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁,

wherein; X₁ is S; X₂ is A or G; X₃ is D; X₄ is K, N, L, Q, M, E, S, T,G, or D; X₅ is L; X₆ is G; X₇ is T, S, N, A, G, or E; X₈ is K, Q, N, orR; X₉ is Y; X₁₀ is V or I; and X₁₁ is S; CDR CDR-L2: (SEQ ID NO: 109)X1-X2-X3-X4-X5-X6-X7,

wherein; X₁ is Q; X₂ is D; X₃ is A, G, W, S, or D; X₄ is K, M, Q, N, L,T, I, or E; X₅ is R; X₆ is P; and X₇ is S; and CDR-L3: (SEQ ID NO: 110)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉,

wherein; X₁ is Q; X₂ is S or A; X₃ is W; X₄ is D; X₅ is R, S, Q, P, A,V, W, or M; X₆ is S, G, I, N, R, or T; X₇ is D or G; X₈ is V, A, P, orE; and X₉ is V.
 95. The binding protein according to claim 94, whereinsaid at least one CDR comprises an amino acid sequence selected from thegroup consisting of: residues 31-35 of SEQ ID NO: 112 (CDR-H1); residues50-66 of SEQ ID NO:112 (CDR-H2); residues 99-108 of SEQ ID NO:112(CDR-H3); residues 23-33 of SEQ ID NO: 113 (CDR-L1); residues 49-55 ofSEQ ID NO: 113 (CDR-L2); residues 88-96 of SEQ ID NO: 113 (CDR-L3);residues 31-35 of SEQ ID NO:316 (CDR-H1); residues 50-66 of SEQ IDNO:316 (CDR-H2); residues 99-108 of SEQ ID NO:316 (CDR-H3); residues31-35 of SEQ ID NO:317 (CDR-H1); residues 50-66 of SEQ ID NO:317(CDR-H2); residues 99-108 of SEQ ID NO:317 (CDR-H3); residues 31-35 ofSEQ ID NO:318 (CDR-H1); residues 50-66 of SEQ ID NO:318 (CDR-H2);residues 99-108 of SEQ ID NO:318 (CDR-H3); residues 31-35 of SEQ IDNO:319 (CDR-H1); residues 50-66 of SEQ ID NO:319 (CDR-H2); residues99-108 of SEQ ID NO:319 (CDR-H3); residues 31-35 of SEQ ID NO:320(CDR-H1); residues 50-66 of SEQ ID NO:320 (CDR-H2); residues 99-108 ofSEQ ID NO:320 (CDR-H3); residues 31-35 of SEQ ID NO:321 (CDR-H1);residues 50-66 of SEQ ID NO:321 (CDR-H2); residues 99-108 of SEQ IDNO:321 (CDR-H3); residues 31-35 of SEQ ID NO:322 (CDR-H1); residues50-66 of SEQ ID NO:322 (CDR-H2); residues 99-108 of SEQ ID NO:322(CDR-H3); residues 31-35 of SEQ ID NO:323 (CDR-H1); residues 50-66 ofSEQ ID NO:323 (CDR-H2); residues 99-108 of SEQ ID NO:323 (CDR-H3);residues 31-35 of SEQ ID NO:324 (CDR-H1); residues 50-66 of SEQ IDNO:324 (CDR-H2); residues 99-108 of SEQ ID NO:324 (CDR-H3); residues31-35 of SEQ ID NO:325 (CDR-H1); residues 50-66 of SEQ ID NO:325(CDR-H2); residues 99-108 of SEQ ID NO:325 (CDR-H3); residues 31-35 ofSEQ ID NO:326 (CDR-H1); residues 50-66 of SEQ ID NO:326 (CDR-H2);residues 99-108 of SEQ ID NO:326 (CDR-H3); residues 23-33 of SEQ IDNO:327 (CDR-L1); residues 49-55 of SEQ ID NO:327 (CDR-L2); residues88-96 of SEQ ID NO:327 (CDR-L3); residues 23-33 of SEQ ID NO:328(CDR-L1); residues 49-55 of SEQ ID NO:328 (CDR-L2); residues 88-96 ofSEQ ID NO:328 (CDR-L3); residues 23-33 of SEQ ID NO:329 (CDR-L1);residues 49-55 of SEQ ID NO:329 (CDR-L2); residues 88-96 of SEQ IDNO:329 (CDR-L3); residues 23-33 of SEQ ID NO:330 (CDR-L1); residues49-55 of SEQ ID NO:330 (CDR-L2); residues 88-96 of SEQ ID NO:330(CDR-L3); residues 23-33 of SEQ ID NO:331 (CDR-L1); residues 49-55 ofSEQ ID NO:331 (CDR-L2); residues 88-96 of SEQ ID NO:331 (CDR-L3);residues 23-33 of SEQ ID NO:332 (CDR-L1); residues 49-55 of SEQ IDNO:332 (CDR-L2); residues 88-96 of SEQ ID NO:332 (CDR-L3); residues23-33 of SEQ ID NO:333 (CDR-L1); residues 49-55 of SEQ ID NO:333(CDR-L2); residues 88-96 of SEQ ID NO:333 (CDR-L3).
 96. The bindingprotein according to claim 95, wherein said binding protein comprises atleast 3 CDRs.
 97. The binding protein according to claim 96, whereinsaid at least 3 CDRs comprises a variable domain CDR set selected fromthe group consisting of: VH A10 CDR Set CDR-H1: residues 31-35 of SEQ IDNO:112 CDR-H2: residues 50-66 of SEQ ID NO: 112 CDR-H3: residues 99-108of SEQ ID NO:112 VL A10 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:113CDR-L2: residues 49-55 of SEQ ID NO:113 CDR-L3: residues 88-96 of SEQ IDNO: 113 VH A10.3 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:316 CDR-H2:residues 50-66 of SEQ ID NO:316 CDR-H3: residues 99-108 of SEQ ID NO:316VL A10.3 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:327 CDR-L2:residues 49-55 of SEQ ID NO:327 CDR-L3: residues 88-96 of SEQ ID NO:327VH A10.K30 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:317 CDR-H2:residues 50-66 of SEQ ID NO:317 CDR-H3: residues 99-108 of SEQ ID NO:317VH A10.K42 CDR Set CDR-H1: residues 31-35 of SEQ ID NO:318 CDR-H2:residues 50-66 of SEQ ID NO:318 CDR-H3: residues 99-108 of SEQ ID NO:318VH A10.9A CDR Set CDR-H1: residues 31-35 of SEQ ID NO:319 CDR-H2:residues 50-66 of SEQ ID NO:319 CDR-H3: residues 99-108 of SEQ ID NO:319VH A10.8A CDR Set CDR-H1: residues 31-35 of SEQ ID NO:320 CDR-H2:residues 50-66 of SEQ ID NO:320 CDR-H3: residues 99-108 of SEQ ID NO:320VH A10.1A CDR Set CDR-H1: residues 31-35 of SEQ ID NO:321 CDR-H2:residues 50-66 of SEQ ID NO:321 CDR-H3: residues 99-108 of SEQ ID NO:321VH A10.5D CDR Set CDR-H1: residues 31-35 of SEQ ID NO:322 CDR-H2:residues 50-66 of SEQ ID NO:322 CDR-H3: residues 99-108 of SEQ ID NO:322VH A10.3A CDR Set CDR-H1: residues 31-35 of SEQ ID NO:323 CDR-H2:residues 50-66 of SEQ ID NO:323 CDR-H3: residues 99-108 of SEQ ID NO:323VL A10.3A CDR Set CDR-L1: residues 23-33 of SEQ ID NO:330 CDR-L2:residues 49-55 of SEQ ID NO:330 CDR-L3: residues 88-96 of SEQ ID NO:330VH A10.6B CDR Set CDR-H1: residues 31-35 of SEQ ID NO:324 CDR-H2:residues 50-66 of SEQ ID NO:324 CDR-H3: residues 99-108 of SEQ ID NO:324VL A10.6B CDR Set CDR-L1: residues 23-33 of SEQ ID NO:331 CDR-L2:residues 49-55 of SEQ ID NO:331 CDR-L3: residues 88-96 of SEQ ID NO:331VH A10.3D CDR Set CDR-H1: residues 31-35 of SEQ ID NO:325 CDR-H2:residues 50-66 of SEQ ID NO:325 CDR-H3: residues 99-108 of SEQ ID NO:325VL A10.3D CDR Set CDR-L1: residues 23-33 of SEQ ID NO:332 CDR-L2:residues 49-55 of SEQ ID NO:332 CDR-L3: residues 88-96 of SEQ ID NO:332VH A10.4C CDR Set CDR-H1: residues 31-35 of SEQ ID NO:326 CDR-H2:residues 50-66 of SEQ ID NO:326 CDR-H3: residues 99)-108 of SEQ IDNO:326 VL A10.4C CDR Set CDR-L1: residues 23-33 of SEQ ID NO:333 CDR-L2:residues 49-55 of SEQ ID NO:333 CDR-L3: residues 88-96 of SEQ ID NO:333VL A10.L45 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:328 CDR-L2:residues 49-55 of SEQ ID NO:328 CDR-L3: residues 88-96 of SEQ ID NO:328VL A10.L73 CDR Set CDR-L1: residues 23-33 of SEQ ID NO:329 CDR-L2:residues 49-55 of SEQ ID NO:329 CDR-L3: residues 88-96 of SEQ ID NO:32998. The binding protein according to claim 97, comprising at least twovariable domain CDR Sets.
 99. The binding protein according to claim 98,wherein said at least two variable domain CDR sets are selected from agroup consisting of: VH A10 CDR Set and VL A10 CDR Set; VH A10.3 CDR Setand VL A10.3 CDR Set; VH A10.3A CDR Set and VL A10.3A Set; VH A10.6B CDRSet and VL A10.6B Set; VH A10.3D CDR Set and VL A10.3D CDR Set; VHA10.4C CDR Set and VL A10.4C CDR Set; VH A10.K30 CDR Set and VL A10.3CDR Set; VH A10.K42 CDR Set and VL A10.3 CDR Set; VH A10.3 CDR Set andVL A10.L45 CDR Set; VH A10.3 CDR Set and VL A10.L73 CDR Set; VH A10.9ACDR Set and VL A10.3 CDR Set; VH A10.8A CDR Set and VL A10.3 CDR Set; VHA10.1A CDR Set and VL A10.3 CDR Set; and VH A10.5D CDR Set and VL A10.3CDR Set.
 100. The binding protein according to any one of claims 94-99,further comprising a human acceptor framework.
 101. The binding proteinaccording to claim 100, wherein said human acceptor framework comprisesan amino acid sequence selected from the group consisting of: heavychain acceptor framework sequences SEQ ID NOS:6-22, heavy chain acceptorsequences SEQ ID NOS:35-62, light chain acceptor sequences SEQ IDNOS:23-34, and light chain acceptor sequences SEQ ID NOS:63-98.
 102. Thebinding protein according to claim 100 or claim 101, wherein said humanacceptor framework comprises at least one framework region amino acidsubstitution, wherein the amino acid sequence of the framework is atleast 65% identical to the sequence of said human acceptor framework andcomprising at least 70 amino acid residues identical to said humanacceptor framework.
 103. The binding protein according to claim 101,wherein said human acceptor framework comprises at least one frameworkregion amino acid substitution at a key residue, said key residueselected from the group consisting of: a residue adjacent to a CDR; aglycosylation site residue; a rare residue; a residue capable ofinteracting with human DLL4 a canonical residue; a contact residuebetween heavy chain variable region and light chain variable region; aresidue within a Vernier zone; and a residue in a region that overlapsbetween a Chothia-defined variable heavy chain CDR1 and a Kabat-definedfirst heavy chain framework.
 104. The binding protein according to claim103, wherein key residue selected from the group consisting of: 2H, 4H,24H, 26H, 27H, 29H, 34H, 35H, 37H, 39H, 44H, 45H, 47H, 48H, 49H, 50H,51H, 58H, 59H, 60H, 63H, 67H, 69H, 71H, 73H, 76H, 78H, 91H, 93H, 94H,2L, 4L, 25L, 29L, 27bL, 33L, 34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L,49L, 55L, 58L, 62L, 64L, 71L, 87L, 89L, 90L, 91L, 94L, 95L.
 105. Thebinding protein according to claim 104, where the binding protein is aconsensus human variable domain.
 106. The binding protein according toclaim 94, where said binding protein comprises at least one variabledomain having an amino acid sequence selected from the group consistingof: SEQ ID NOS:112, 113, 316, 327, 317, 318, 319, 320, 321, 322, 323,330, 324, 331, 325, 332, 326, 333, 328, and
 329. 107. The bindingprotein according to claim 106, wherein said binding protein comprisestwo variable domains, wherein said two variable domains have amino acidsequences selected from the group consisting of: SEQ ID NOS:112 and 113,SEQ ID NOS:316 and 327, SEQ ID NOS:323 and 330, SEQ ID NOS:324 and 331,SEQ ID NOS:325 and 332, and SEQ ID NOS:326 and
 333. 108. The bindingprotein according to claim 104, wherein said binding protein comprisesat least one variable domain having an amino acid sequence selected fromthe group consisting of: SEQ ID NOS: 112, 113, 316, 327, 317, 318, 319,320, 321, 322, 323, 330, 324, 331, 325, 332, 326, 333, 328, and 329.109. The binding protein according to claim 95 wherein said antigenbinding domain comprises a V_(H).
 110. The binding protein according toclaim 109 wherein said V_(H) comprises an amino acid sequence selectedfrom the group consisting of: SEQ ID NOS:112, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, and
 326. 111. The binding protein according toclaim 95 wherein said antigen binding domain comprises a V_(L).
 112. Thebinding protein according to claim 111 wherein said V_(L) comprises anamino acid sequence selected from the group consisting of: SEQ IDNOS:113, 327, 328, 329, 330, 331, 332, and
 333. 113. The binding proteinaccording to claim 95 wherein said antigen binding domain comprises aV_(H) and a V_(L).
 114. The binding protein according to claim 112further comprising a V_(H) wherein said V_(H) comprises an amino acidsequence selected from the group consisting of: SEQ ID NOS: SEQ IDNOS:112, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, and
 326. 115.The binding protein according to claim 20 wherein said V_(L) comprisesan amino acid sequence selected from the group consisting of: SEQ IDNOS: 113, 327, 328, 329, 330, 331, 332, and
 333. 116. The bindingprotein according to claim 95, further comprising a heavy chainimmunoglobulin constant domain selected from the group consisting of: ahuman IgM constant domain; a human IgG1 constant domain; a human IgG2constant domain; a human IgG3 constant domain; a human IgG4 constantdomain: a human IgE constant domain and a human IgA constant domain.117. The binding protein according to claim 117 wherein said heavy chainimmunoglobulin constant region domain is a human IgG constant domain.118. The binding protein according to claim 117 wherein said human IgG1constant domain comprises amino acid sequence selected from the groupconsisting of SEQ ID NO:2 and SEQ ID NO:3.
 119. The binding proteinaccording to claim 95, further comprising a light chain immunoglobulinconstant domain selected from the group consisting of: a human Ig kappaconstant domain and a human Ig lambda constant domain.
 120. The bindingprotein according to claim 119 wherein said light chain immunoglobulinconstant region domain is a human Ig kappa constant domain comprisingamino acid sequence SEQ ID NO:4.
 121. The binding protein according toclaim 119 wherein said light chain immunoglobulin constant region domainis a human Ig lambda constant domain comprising amino acid sequence SEQID NO:5.
 122. The binding protein according to claim 94 wherein saidbinding protein is selected from the group consisting of: animmunoglobulin molecule, an scFv, a monoclonal antibody, a humanantibody, a chimeric antibody, a humanized antibody, a single domainantibody, a Fab fragment, a Fab′ fragment, an F(ab′)₂, an Fv, adisulfide linked Fv, a single domain antibody, a diabody, amultispecific antibody, a bispecific antibody, and a dual specificantibody.
 123. The binding protein according to claim 122 wherein saidbinding protein is a human antibody.
 124. A binding protein capable ofbinding human DLL-4, said binding protein comprising: an Ig constantheavy region having an amino acid sequence selected from the groupconsisting of SEQ ID NO:2 and SEQ ID NO:3; an Ig constant light regionhaving an amino acid sequence selected from the group consisting of SEQID NO:4 and SEQ ID NO:5; an Ig variable heavy region having an aminoacid sequence selected from the group consisting: SEQ ID NOS: SEQ IDNOS: 112, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, and 326; andan Ig variable light region having an amino acid sequence selected fromthe group consisting: SEQ ID NOS: 113, 327, 328, 329, 330, 331, 332, and333.
 125. The binding protein according to claim 124, wherein the Igconstant light region is SEQ ID NO:5.
 126. The binding protein accordingto any one of claims 94-125, wherein the binding protein is capable ofblocking DLL4 interaction with a Notch protein selected from the groupconsisting of Notch-1, Notch-2, Notch-3, Notch-4, and combinationsthereof.
 127. The binding protein according to claim 126, wherein thebinding protein is capable of blocking DLL4 interaction with Notch-1 andNotch-4.
 128. The binding protein according to claim 126, wherein thebinding protein is capable of blocking DLL4 interaction with Notch-1.129. The binding protein according to claim 126, wherein the bindingprotein is capable of blocking DLL4 interaction with Notch-4.
 130. Thebinding protein according to any one of claims 94-125, wherein thebinding protein is capable of modulating a biological function of DLL4.131. The binding protein according to any one of claims 94-125, whereinsaid binding protein is capable of neutralizing a DLL4.
 132. Theneutralizing binding protein according to claim 131 wherein said DLL4 isselected from the group consisting of: human DLL4, mouse DLL4,cynomolgus DLL4, and rat DLL4.
 133. The neutralizing binding proteinaccording to claim 131 wherein said neutralizing binding proteindiminishes the ability of DLL4 to bind to its receptor.
 134. Theneutralizing binding protein according to claim 131 wherein saidneutralizing binding protein is capable of reducing normal angiogenesis.135. The neutralizing binding protein according to claim 131, whereinsaid neutralizing binding protein has a dissociation constant (K_(D))selected from the group consisting of: at most about 10⁻⁷ M; at mostabout 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at most about10⁻¹¹ M; at most about 10⁻¹² M; and at most 10⁻¹³ M.
 136. Theneutralizing binding protein according to claim 131, wherein saidneutralizing binding protein has an on rate selected from the groupconsisting of; at least about 10²M⁻¹s⁻¹; at least about 10³M⁻¹ s⁻¹; atleast about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; and at least about10⁶M⁻¹s⁻¹.
 137. The neutralizing binding protein according to claim 131,wherein said neutralizing binding protein has an off rate selected fromthe group consisting of; at most about 10⁻³s⁻¹; at most about 10⁻⁴s⁻¹;at most about 10⁻⁵s⁻¹; and at most about 10⁻⁶s⁻¹.
 138. A labeled bindingprotein comprising a binding protein of any one of claims 94-137,wherein said binding protein is conjugated to a detectable label. 139.The labeled binding protein of claim 138, wherein the detectable labelis selected from the group consisting of a radiolabel, an enzyme, afluorescent label, a luminescent label, a bioluminescent label, amagnetic label, and biotin.
 140. The labeled binding protein of claim139, wherein said label is a radiolabel selected from the groupconsisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho,and ¹⁵³Sm.
 141. An antibody construct comprising a binding proteindescribed in any of claims 94-137 and further comprising a linkerpolypeptide or an immunoglobulin constant domain.
 142. The antibodyconstruct according to claim 141, selected from the group consisting of:an immunoglobulin molecules, a monoclonal antibody, a chimeric antibody,a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)₂,a Fv, a disulfide linked Fv, a scFv, a single domain antibody, adiabody, a multispecific antibody, a dual specific antibody, and abispecific antibody.
 143. The antibody construct according to claim 141,wherein said antibody construct comprises a heavy chain immunoglobulinconstant domain selected from the group consisting of: a human IgMconstant domain, a human IgG1 constant domain, a human IgG2 constantdomain, a human IgG3 constant domain, a human IgG4 constant domain, ahuman IgE constant domain, a human IgA constant domain, and a IgGconstant domain variant with one or more mutations altering bindingstrength to Fc neonatal receptor, Fc gamma receptors, or C1 q.
 144. Theantibody construct according to claim 141, comprising an immunoglobulinconstant domain having an amino acid sequence selected from consistingof: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinationsthereof.
 145. An antibody conjugate comprising an antibody construct asdescribed in claim 141, wherein said antibody construct is conjugated toa therapeutic or cytotoxic agent.
 146. The antibody conjugate of claim145, wherein said therapeutic or cytotoxic agent is selected from thegroup consisting of: an anti-metabolite, an alkylating agent, anantibiotic, a growth factor, a cytokine, an anti-angiogenic agent, ananti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.147. An isolated nucleic acid encoding a polypeptide selected from thegroup consisting of: a polypeptide comprising a heavy chain variabledomain, wherein the heavy chain variable domain comprises one or more ofCDR-H1, a CDR-H2, or a CDR-H3 as described in claim 94; a polypeptidecomprising a light chain variable domain, wherein the light chainvariable domain comprises one or more of CDR-L1, a CDR-L2, or a CDR-L3as described in claim 94; or a combination of both polypeptides.
 148. Avector comprising an isolated nucleic acid according to claim
 147. 149.The vector of claim 148 wherein said vector is selected from the groupconsisting of: pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pHybE and pBJ.
 150. Ahost cell comprising a vector according to any one of claims 148 and149.
 151. The host cell according to claim 150, wherein said host cellis a prokaryotic cell.
 152. The host cell according to claim 151,wherein said host cell is Escherichia coli.
 153. The host cell accordingto claim 152, wherein said host cell is a eukaryotic cell.
 154. The hostcell according to claim 153, wherein said eukaryotic cell is selectedfrom the group consisting of: a protist cell, an animal cell, a plantcell, and a fungal cell.
 155. The host cell according to claim 154,wherein said eukaryotic cell is an animal cell selected from the groupconsisting of: a mammalian cell, an avian cell, and an insect cell. 156.The host cell according to claim 155, wherein said mammalian cell is aCHO cell.
 157. The host cell according to claim 155, wherein saidmammalian cell is a COS cell.
 158. The host cell according to claim 154,wherein said fungal cell is Saccharomyces cerevisiae.
 159. The host cellaccording to claim 155, wherein said insect cell is an Sf9 cell.
 160. Amethod of producing a binding protein that binds human DLL4, comprisingculturing the host cell of any one of claims 150-159 in a culture mediumunder conditions sufficient to produce a binding protein that bindshuman DLL4.
 161. A binding protein produced according to the method ofclaim
 160. 162. A crystallized binding protein comprising a bindingprotein according to any one of claims 94-137, wherein said bindingprotein exists as a crystal.
 163. The crystallized binding proteinaccording to claim 162, wherein said crystal is a carrier-freepharmaceutical controlled release crystal.
 164. The crystallized bindingprotein according to claim 162, wherein said binding protein has agreater half life in vivo than the soluble counterpart of said bindingprotein.
 165. The crystallized binding protein according to claim 162,wherein said binding protein retains biological activity.
 166. Acomposition for the release of a binding protein said compositioncomprising: (a) a formulation, wherein said formulation comprises acrystallized binding protein, according to any one of claims 162-165,and an ingredient; and (b) at least one polymeric carrier.
 167. Thecomposition according to claim 166, wherein said polymeric carrier is apolymer selected from one or more of the group consisting of poly(acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid),poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutyrate), poly(caprolactone), poly (dioxanone), poly (ethylene glycol), poly((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolyeaccharides, blends and copolymers thereof.
 168. The compositionaccording to claim 166, wherein said ingredient is selected from thegroup consisting of albumin, sucrose, trehalose, lactitol, gelatin,hydroxypropyl-43-cyclodextrin, methoxypolyethylene glycol andpolyethylene glycol.
 169. A method for treating a mammal comprising thestep of administering to the mammal an effective amount of thecomposition according to claim
 166. 170. A pharmaceutical compositioncomprising the binding protein of any one of claims 94-137, and apharmaceutically acceptable carrier.
 172. The pharmaceutical compositionof claim 170 which further comprises at least one additional therapeuticagent for treating a disorder in which DLL4 activity is detrimental.173. The pharmaceutical composition of claim 172, wherein saidadditional agent is selected from the group consisting of: angiogenesisinhibitors; kinase inhibitors; co-stimulation molecule blockers;adhesion molecule blockers: anti-cytokine antibody or functionalfragment thereof; methotrexate; corticosteroids; cyclosporine;rapamycin; FK506; and non-steroidal anti-inflammatory agents.
 174. Amethod for reducing human DLL4 activity comprising contacting human DLL4with the binding protein of any one of claims 94-137 such that humanDLL4 activity is reduced.
 175. A method for reducing human DLL4 activityin a human subject suffering from a disorder in which DLL4 activity isdetrimental, comprising administering to the human subject the bindingprotein of any one of claims 94-137 such that human IL-17 activity inthe human subject is reduced.
 176. A method for treating a subject for adisease or a disorder in which DLL4 activity is detrimental byadministering to the subject the binding protein of any one of claims94-137 such that treatment is achieved.
 177. The method of claim 176,wherein said disorder is selected from the group consisting of: breastcancer, colon cancer, rectal cancer, lung cancer, oropharynx cancer,hypopharynx cancer, esophageal cancer, stomach cancer, pancreas cancer,liver cancer, gallbladder cancer, bile duct cancer, small intestinecancer, urinary tract cancer, female genital tract cancer, male genitaltract cancer, endocrine gland cancer, skin cancer, hemangiomas,melanomas, sarcomas, brain tumor, nerve cancer, eye tumor, meningescancer, solid tumors from hematopoietic malignancies, tumor metastases,ocular neovascularization, edema, rheumatoid arthritis, multiplesclerosis, atheroscleorotic plaques, Crohn's disease, inflammatory boweldisease, refractory ascites, psoriasis, sarcoidosis, arterialarteriosclerosis, sepsis, peptic ulcers, burns, and pancreatitis,polycystic ovarian disease (POD), endometriosis, uterine fibroids,benign prostate hypertrophy, and other angiogenesis independent anddependent diseases characterized by abberant DLL4 activity.
 178. Themethod according to claim 177, wherein the disorder is a primary andmetastatic cancer.
 179. The method according to claim 177, wherein theurinary tract cancer is selected from the group consisting of renalcancer, bladder cancer, and urothelium cancer.
 180. The method accordingto claim 177, wherein the female genital tract cancer is selected fromthe group consisting of cervical cancer, uterine cancer, ovarian cancer,choriocarcinoma, and gestational trophoblastic disease.
 181. The methodaccording to claim 177, wherein the male genital tract cancer isselected from the group consisting of prostate cancer, seminal vesiclescancer, testicular cancer, and germ cell tumor.
 182. The methodaccording to claim 177, wherein the endocrine gland cancer is selectedfrom the group consisting of thyroid cancer, adrenal cancer, andpituitary gland cancer.
 183. The method according to claim 177, whereinthe sarcoma is selected from the group consisting of a bone sarcoma, asoft tissue sarcoma, and Kaposi's sarcoma.
 184. The method according toclaim 177, wherein the meninges cancer is selected from the groupconsisting of an astrocytoma, a glioma, a glioblastoma, aretinoblastoma, a neuroma, a neuroblastoma, a Schwannoma, and ameningiomas.
 185. The method according to claim 177, wherein the solidtumor from a hematopoietic malignancy is a leukemia, a Hodgkin'sleukemia, a non-Hodgkin's leukemia, a lymphoma, a Hodgkin's lymphoma,and a non-Hodgkin's lymphomas.
 186. The method according to claim 177,wherein the ocular neovascularization is selected from the groupconsisting of diabetic blindness, a retinopathy, an age-induced maculardegeneration, and a rubeosis.
 187. A method of treating a patientsuffering from a disorder in which DLL4 is detrimental comprising thestep of administering the binding protein of any one of claims 94-137before, concurrent with, or after the administration of a second agent,wherein the second agent is selected from the group consisting of anantibody or fragment thereof capable of binding human VEGFR2:methotrexate: an antibody, or fragment thereof, capable of binding humanTNF: corticosteroids, cyclosporine, rapamycin, FK506, and non-steroidalanti-inflammatory agents.